Formulations for oral delivery of adsorbents in the gut

ABSTRACT

The invention relates to a formulation for the delayed and controlled delivery of an adsorbent into the lower intestine of mammals. The formulation includes a carrageenan and an adsorbent, such as activated charcoal. The invention further relates to uses of this formulation, in particular to pharmaceutical uses. In one embodiment, the formulation is used to eliminate or reduce the side effects in the intestine, in particular in the colon, of pharmaceutical agents that are administered as a treatment for a disorder, but that have side effects when they reach the late ileum, the caecum or the colon.

RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priorityunder 35 U.S.C. § 120 to U.S. patent application Ser. No. 13/580,144,filed on Oct. 31, 2012, which was the national stage entry under 35U.S.C. § 371 of PCT/EP2011/052682, filed on Feb. 23, 2011, which itselfclaims the benefit under 35 U.S.C. 119(e) of European Patent ApplicationNo. 10305179.3, filed on Feb. 23, 2010, the contents of each of whichare incorporated herein by reference in their entireties for allpurposes.

BACKGROUND OF THE INVENTION

When antibiotics are administered, either orally or parenterally, asignificant fraction of the administered dose reaches the late ileum orcolon in an active form and comes into close contact with the bacterialpopulation that is present in the colon. The alarming consequences ofthis have been known for years and constitute the subject of a ECDC/EMEAJoint Technical Report called “The bacterial challenge: time to react, Acall to narrow the gap between multidrug-resistant bacteria in the EUand the development of new antibacterial agents” published in September2009. The residual antibiotic exerts a selective pressure on thebacteria present in the colon and provokes the emergence and developmentof bacteria resistant to the antibiotic. Because the geneticdeterminants of resistance to various antibiotics are often physicallylinked on mobile genetic elements such as plasmids and transposons, thetreatment with a single antibiotic often selects for the simultaneouspresence of several antibiotic resistance genes, thus explaining howmulti-antibiotic resistance can emerge very fast.

As a result of this process, the patient or the animal that has receivedan antibiotic treatment becomes very rapidly and strongly colonized byantibiotic-resistant bacteria. This can result in complicated furtherinfections by resistant bacteria as well as the dissemination ofresistance to other bacteria, and ultimately the environment.

It is now widely accepted that the selection and dissemination of suchresistant bacteria is a major factor that increases significantly thedissemination of bacterial resistance to antibiotics both in thecommunity and in hospitals. Levels of bacterial resistance are currentlyextremely high and increasing year after year becoming a major publichealth problem worldwide that could lead to major outbreaks ofinfections very difficult to treat with available antibiotics either inhumans or in animals.

Besides producing antibiotic-resistant bacteria, antibiotics that reachthe colon in active form will also profoundly alter the composition ofthe commensal flora and eliminate susceptible bacterial species. Amongthose bacteria, susceptible anaerobic bacteria can be eliminated; theyare known to play a major physiological role in the intestine of normalsubjects and animals. For example, they act to prevent colonization byexogenous potentially pathogenic microorganisms such as Clostridiumdifficile and/or Candida sp, and/or multiresistant exogenous bacteriasuch as Vancomycin-resistant enterococci. It is therefore essential toprevent elimination of such useful bacteria to prevent adverse effectsof antibiotics, which can lead to the appearance of pathologic signs andsymptoms, such as post-antibiotic diarrhea or the more severe forms ofpseudomembranous colitis, Candida genital infections, particularly inwomen, or antibiotic-resistant systemic infections in hospitalizedpatients, particularly those in intensive care.

One way to prevent such adverse effects of antibiotic treatments is toeliminate residual antibiotics that arrive in the caecum and colon; inthe recent years, there have been two different approaches to achievethis goal. One has been the delivery to the gut of enzymes thatspecifically degrade antibiotics (such as those described inUS20050249716). Alternatively, the formulation of an adsorbent for asite-specific intestinal delivery has been proposed in applicationsWO2006/122835 and WO2007/132022. The adsorbent would act by sequesteringthe antibiotic before it can affect susceptible bacteria in the caecumand colon. This approach would enable to broaden the spectra ofantibiotics that may be eliminated as compared to previous approachesbased on antibiotic-specific enzymes. Adsorbents, and in particularactivated charcoal, are very challenging products to formulate becauseof their physicochemical properties such as low density, hydrophobicity,wetting properties, etc. Attempting to formulate activated charcoal foran intestinal site-specific delivery of an oral dose is not possibleusing conventional direct compression because of the very low cohesiveproperties of activated charcoal. Even simple wet granulation andcompression lead to tablets exhibiting poor adsorption properties andpoor disintegration profile. Enzyme-based delivery systems have beenproposed to overcome these problems. These systems are based on theirdegradation and subsequent release of their content in the colon as aresult of the action of colonic enzymes on a polymer encapsulating theadsorbent. A representative system implements pectin beads specificallydegraded by pectinolytic enzymes which are produced in the colon of manymammals by bacteria of the commensal flora (such as those described inWO2006/122835). However, this system presents limitations such as lowadsorbent content and difficulties in up scaling the production ofpectin beads. Also, variability in the amount of pectinolytic enzymespresent in the colon has led to variability in the delivery of theadsorbent. Solid dosage forms, either in single dosage form such astablet or in a multi-dispersed pellet formulation, have also beenproposed, with an excellent yield and adsorbent content (WO2007/132022).However, even though formulations could be made in a straightforwardmanner, their disintegration properties and the adsorption efficiency ofthe released adsorbent could be improved in a more satisfactory manner.

It would be advantageous to develop a formulation appropriate for thedelayed release of an adsorbent in the later parts of thegastrointestinal tract yet preserving as much as possible the adsorptioncharacteristics of the adsorbent. It would also be advantageous toproduce a formulation with an improved adsorbent-release profile, with arelease of the adsorbent at a place and time in the gastrointestinaltract where no more antibiotic is absorbed. This would prevent anyinteraction of the adsorbent with the normal absorption process ofantibiotics, or any other pharmaceutical product, when givensimultaneously by oral route.

Such formulation would be advantageous in removing residual antibioticsand/or their active metabolites from the intestinal tract while beingable to be co-administered with large number of antibiotics and toreduce unwanted antibiotic-associated side-effects such as diarrhea,abdominal pain, and bacterial resistance to antibiotics. It would alsobe advantageous to have formulations that provide a specific release ofan adsorbent in the lower part of the gastrointestinal tract,specifically in the late ileum, the caecum, or the colon.

Such formulation would also be advantageous in reducing or eliminatingthe side effects of pharmaceutical agents or metabolites thereof in thelate ileum, caecum and colon. Such pharmaceutical agents are for exampleagents which are administered to treat a disease state, but which haveside effects when they reach the lower part of the gastroinstestinaltract, specifically in the late ileum, the caecum, or the colon.Representative, non limiting, examples of such pharmaceutical agentsinclude irinotecan and its metabolite SN-38, diacerhein, Pancrelipase(such as Pancrease, Creon, Zenpep), Phosphodiesterase 4 inhibitors usedin the treatment of chronic obstructive pulmonary disease such asRoflumilast or Cilomilast, or anti-mitotic and anti-inflammatory drugssuch as colchicine.

Furthermore, such formulation would be advantageous in the treatment ofdisease states characterized by the accumulation of substances in thelower part of the gastroinstestinal tract, this accumulation beingresponsible for the development of a number of pathological conditions.For example, the formulation can be useful for the treatment ofconditions such as, but not limited to, hepatic encephalopathy,irritable bowel syndrome, chronic renal disease, C. difficile associateddiarrhea or antibiotic associated diarrhea. Representative substanceswhich can be adsorbed by the formulation disclosed herein include, butare not limited to, ammonia, indoles, advanced glycation end products(AGEs) and certain bacterial toxins.

More generally, the formulation of the invention can be used in thetreatment of a condition, either pathological or not, which is caused,maintained and/or enhanced by the presence, or the presence in excessquantities, of certain substances in the lower part of thegastrointestinal tract, specifically in the late ileum, the caecum, orthe colon.

The present invention provides such formulations and methods ofpreparation and use thereof.

SUMMARY OF THE INVENTION

Formulations useful for delivering an adsorbent to the late ileum, thecaecum or the colon are provided. In one embodiment, a compositioncomprising a mixture of an adsorbent with carrageenan, preferably in theform of a pellet, is provided. In one aspect of this embodiment, theadsorbent is activated charcoal, and in another aspect of thisembodiment, the carrageenan is a kappa-carrageenan. The amount ofcarrageenan is typically in the range of between 5% and 25%, morepreferably between 10% and 20%, by weight of the mixture.

The composition comprising the mixture can be used to form a core. Inone embodiment, the core is provided with a layer of a coating such thatthe adsorbent is released from the formulation in the lower part of theintestine, i.e in the late ileum, caecum and/or colon. Representativecoatings allowing release at the desired part of the intestine includepH-dependent enterosoluble polymers, materials that are specificallydegraded in the colonic environment by the action of microorganismsand/or the reductive environment found there (e.g. azopolymers anddisulphide polymers, polysaccharides, in particular amylose or pectin(e.g. pectin crosslinked with divalent cations such as calcium pectinateor zinc pectinate), chondroitin sulphate and guar gum). RepresentativepH-dependent enterosoluble polymers include cellulose acetatetrimellitate (CAT), cellulose acetate phthalate (CAP), anioniccopolymers based on methylacrylate, methylmethacrylate and methacrylicacid, hydroxypropyl methylcellulose phthalate (HPMCP),hydroxypropylmethylcellulose acetate succinate (HPMCAS), methacrylicacid and ethyl acrylate copolymers, methacrylic acid and ethyl acrylatecopolymer, methacrylic acid and methyl methacrylate copolymers (1:1ratio), methacrylic acid and methyl methacrylate copolymers (1:2 ratio),Polyvinyl acetate phthalate (PVAP) and Shellac resins. Particularlypreferred polymers include shellac, anionic copolymers based on methylacrylate, methyl methacrylate and methacrylic acid, and methacrylic acidand methyl methacrylate copolymers (1:2 ratio). Ideally, the polymerdissolves at a pH equal to 6.0 and above, preferably 6.5 and above.

In another embodiment, a further coating is provided between the coreand the external pH-dependent layer. The intermediate coating can beformed from a variety of polymers, including pH-dependent polymers,pH-independent water soluble polymers, pH-independent insolublepolymers, and mixtures thereof.

Representative pH-dependent polymers include shellac type polymers,anionic copolymers based on methylacrylate, methylmethacrylate andmethacrylic acid, methacrylic acid and ethyl acrylate copolymer,hydroxypropyl methylcellulose phthalate (HPMCP), andhydroxypropylmethylcellulose acetate succinate (HPMCAS),

Representative pH-independent water soluble polymers include PVP or highmolecular weight cellulose polymers such as hydroxypropylmethylcellulose(HPMC) or hydroxypropylcellulose (HPC).

Representative pH-independent insoluble polymers include ethylcellulosepolymers or ethyl acrylate methyl methacrylate copolymer.

In one aspect of this embodiment, the polymer layer that dissolves in apH-independent manner includes at least one cellulose-derivativeselected from the group consisting of hydroxypropylcellulose orethylcellulose. In another aspect of this embodiment, the polymer layerthat dissolves in a pH-independent manner is made of a 1:9 to 9:1,preferably 2:8 to 3:7, mixture of methacrylic acid and ethyl acrylatecopolymer and ethyl acrylate methyl methacrylate copolymer.

The formulations can be used to eliminate or reduce the side effects inthe intestine, in particular in the colon, of pharmaceutical agents. Itaims in particular at eliminating or reducing the side effect ofpharmaceutical agents that are administered as a treatment for adisorder, but that have side effects when they reach the late ileum, thecaecum or the colon. For example, the formulations can eliminate orreduce the antibiotic-associated adverse effects of antibiotic agents,eliminate diarrhea, or eliminate the emergence of antibiotic resistance.The formulations can also eliminate a wide variety of pharmaceuticalagents such as, but not only, those mentioned in the following detaileddescription. The formulations can be administered simultaneously with anantibiotic or another pharmaceutical agent.

The formulations can also eliminate or reduce the effects of bacterialor fungal toxins, such as mycotoxins, endotoxins or enterotoxins, orthose produced by Clostridium difficile in the intestine and/or thecolon.

The formulations can also reduce flatulence, stool smell, halitosis orfood intolerance, in particular in a pet or in a farm animal.

Methods of preparing the formulations are also disclosed.

Further object and applications will become apparent in the followingdetailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: kinetics of levofloxacin adsorption by NFAC (non-formulatedactivated charcoal) in simulated colonic fluid.

FIG. 2: calibration of the microbiological assay for ciprofloxacin:relationship between Log 10 ciprofloxacin concentration and growthinhibition diameter.

FIG. 3: adsorption of ciprofloxacin on activated charcoal measured bythe microbiological assay.

FIG. 4: kinetics of levofloxacin adsorption by NFAC and DCP(deformulated coated pellets) in piglet caecal medium.

FIG. 5: desorption of levofloxacin from activated charcoal at variouspHs. Desorption experiments were respectively performed at pH 4.0 (A),7.0 (B) and 10.0 (C). The mean of triplicate determinations±SD is shownfor each data point.

FIG. 6: In vitro comparison of adsorption kinetics of levofloxacin onNFAC and formulated charcoal at two charcoal/levofloxacin ratios.

FIG. 7: BioDis profile ciprofloxacin adsorption onto charcoal releasedfrom FS30D-coated pellets.

FIG. 8: Comparison of coating thicknesses on BioDis profiles of adsorbedciprofloxacin onto charcoal released from pellets with a L30D55/NE30Dsubcoat and a FS30D coating.

FIG. 9: Comparison of FS30D, Aqoat or Shellac coatings on thedissolution profile of different pellet types in simulated ileal medium,pH7.5 (as measured by ciprofloxacine adsorption).

FIG. 10: Comparison of ethylcellulose coating thicknesses on thedissolution profile of pellets in simulated ileal medium, pH7.5 (asmeasured by ciprofloxacine adsorption).

FIG. 11A: Adsorption kinetics of irinotecan on activated charcoal insimulated ileum media, pH7.5.

FIG. 11B: Adsorption kinetic of SN38 on activated charcoal in 1 mM NaOH,pH 12.

FIG. 11C: Adsorption kinetics of irinotecan on activated charcoal incaecal media of piglet spiked with a mixture of SN38 and irinotecan.

FIG. 11D: Adsorption kinetics of SN38 on deformulated coated pellets incaecal media of piglet spiked with a mixture of SN38 and irinotecan.

FIG. 12: In vivo performance of targeted-release activated charcoal inreducing the emergence of bacterial resistance to antibiotic—studydesign.

FIG. 13: Average evolution of faecal ciprofloxacin concentrations bygroup (n1=6, n2=11, n3=12). In this graph, we represent also for eachgroup the 95% confidence interval defined as [mean-1.96*SEM;mean+1.96*SEM] where SEM is the standard error of the mean.

FIG. 14: Average evolution of plasma ciprofloxacin concentrations(ng/mL) by group (n2=n3=12). In this graph, we represent also for eachgroup the 95% confidence interval defined as [mean-1.96*SEM;mean+1.96*SEM] where SEM is the standard error of the mean.

FIG. 15: Resistant bacterial counts: mean of corrected individualAUCciproD1-D8 by treatment group represented by the shaded area betweenthe mean curve of log 10 of ciprofloxacin resistant bacterial countscorrected from the baseline, and the X=0 axis from Day 1 to Day 8 (n1=6,n2=11, n3=12)

FIG. 16: Adsorption kinetics of Creon on activated charcoal in bufferedmedia, pH7.5.

DETAILED DESCRIPTION

The invention relates to a formulation including a carrageenan and anadsorbent. The formulation is suitable for oral administration of anadsorbent and delivery of said adsorbent in the lower part of theintestine, i.e. in the late ileum, the caecum and/or the colon. In oneembodiment, the carrageenan and adsorbent are present as a mixture,which mixture can be compressed to form a core (the core being furtherherein referred to as a particle or pellet).

The core can be coated with one or more coating layers, and the coatedor uncoated cores can be used to form a drug delivery vehicle, such as atablet, capsule, pill, and the like.

The formulations of the invention are solid dosage forms useful fordelivering an adsorbent to a desired part of the intestine,advantageously in the late ileum, the caecum or the colon. The externaland/or intermediate coatings are in particular provided to minimize(preferably to totally prevent) the impact of the adsorbent on thenormal absorption process of a therapeutic agent (for example, anantibiotic) by the host organism when said therapeutic agent isadministered orally along with the formulation according to theinvention. In addition or alternatively, the adsorbent thus formulatedis prevented from non-specifically adsorbing material present in thegastrointestinal tract all the way to the terminal part of the smallintestine. This results in the release of a non saturated adsorbent,fully or almost fully efficient adsorbent in the specific part of theintestine where its action is needed.

Methods of preparing the formulations, and methods of treatment usingthe formulations, are also disclosed. The individual components of theformulations are described in detail below.

Antibiotics

The term “antibiotic” denotes a substance that kills or inhibits thegrowth of microorganisms such as bacteria, fungi, or protozoans.Representative non limiting antibiotics that can be adsorbed accordingto the invention include Beta-lactams such as Amoxicillin, Ampicillin,Piperacillin, Cephalexin, Cefixime, Ceftazidime, Cefuroxime,Ceftriaxone, Cefotaxime, Ceftiofur, Cefdinir, Cefpodoxime, Cefpirome,Cefquinome, Cefepime, Ceftobiprole, Ceftarolime, Ceftiofur, Imipenem,Ertapenem, Doripenem, Meropenem and beta-lactmase inhibitors such asClavulanate, Sulbactam or Tazobactam either alone, or given incombination with other beta-lactam antibiotics; Tetracyclines such asChlortetracycline, Oxytetracycline, Tetracycline, Doxycycline orMinocycline; Macrolides such as Tylosine, Erythromycin, Azithromycin,Clarithromycin, Roxithromycin, Telithromycin, Josamycin, Oleandomycin,Spiramycin, Clindamycin, Lincomycin, Quinupristin or Dalfopristin;Fluoroquinolones such as Nalidixic acid, Ciprofloxacine, Norfloxacin,Ofloxacin, Levofloxacin, Moxifloxacin, Enrofloxacin, Sarafloxacin orMarbofloxacin; Sulfonamides such as Sulfamethoxazole, Sulfadiazine orSulfathiazole; the dihydroflate reductase inhibitor Trimetoprim; theoxazolodinone antibiotic Linezolid: or other antibiotics such asFlorfenicol, Tiamulin or Tigecycline.

Adsorbents

Examples of suitable adsorbents include activated charcoal, clays,including bentonite, kaolin, montmorrillonite, attapulgite, halloysite,laponite, and the like, silica, including colloidal silica (Ludox® AS-40for example), mesoporous silica (MCM41), fumed silica, zeolites and thelike, talc, cholesteramine and the like, polystyrene sulfonates and thelike, mono and polysulfonated resins, and any other resins of interestsuch as those used for bacteriologic testing such as BACTEC® resins.Among these adsorbents, it can be preferred to use those ofpharmaceutical grade, such as activated charcoal USP (Merck, France orother sources), kaolin (VWR, France), attapulgite (Lavollee, France),bentonite (Acros Organics, France), Talc USP (VWR, France).

The amount of adsorbent to produce a single dosage form may varydepending upon the host being treated and the overall capacity andselectivity of the adsorbent towards the antibiotic(s). The amount ofadsorbent to produce a single dosage form will generally be that amountof the compound which produces a desired effect. The desired effect maybe a therapeutic effect, for example a therapeutically significantdecrease in the amount of the antibiotic, metabolite thereof, bacterialtoxin, or other compound which causes adverse effects in the terminalparts of the gut, in particular in the colon, as compared to when theformulation is not administered.

The amount of the adsorbent will range from about 1% to about 99% byweight of the total pellet, preferably from about 50% to about 95%, mostpreferably from about 65% to about 95%, in particular from about 80% toabout 95% by weight of the core formulation.

In a particular embodiment, activated charcoal is used. In one aspect ofthis embodiment, activated charcoal preferentially has a specific areaabove 1500 m²/g, preferentially above 1600 m²/g and best above 1800m²/g.

Carrageenan

Carrageenan is a naturally-occurring family of linear sulphatedpolysaccharides which are extracted from red seaweeds. It is a highmolecular weight polysaccharide made up of repeating galactose and3,6-anhydrogalactose (3,6-AG) units, both sulfated and non-sulfated. Theunits are joined by alternating alpha 1-3 and beta 1-4 glycosidiclinkages. Three basic types of carrageenan are available commercially,i.e. kappa, iota, and lambda carrageenans, which differ by the numberand position of the ester sulfate groups on the galactose units.

In one embodiment, the carrageenan can be selected from kappa, iota andlamba carrageenans, and mixtures thereof. In one aspect of thisembodiment, the adsorbent is mixed with kappa-carrageenan. In aparticular embodiment, the mixture comprises activated charcoal andkappa-carrageenan.

Preferably, the amount of carrageenan is between about 15% and about25%, more preferably between about 10% and about 20%, by weight of themixture of the adsorbent with the carrageenan. According to a specificembodiment of the invention, the amount of carrageenan is about 15% byweight of the mixture. For example, the mixture may contain 85% of anadsorbent and 15% of carrageenan, by weight of the total mixture. Thepossibility of formulating such important amounts of adsorbent withcarrageenan was unexpected, and allows delivery of large amounts ofadsorbent, preferably of activated charcoal, in the desired part of thegut.

According to a particular embodiment of the invention, a mixture ofactivated charcoal and carrageenan is provided with the weight ratioindicated above.

The core (or pellet) may be produced by any suitable means known to theskilled artisan. In particular, granulation techniques are adapted toproduce said core. For example, the core may be obtained by mixing theadsorbent and the carrageenan in the ratio indicated above, adding asolvent such as water to proceed to wet granulation, followed byextrusion spheronization or one-pot pelletization. Any remaining watercan be removed, for example, by drying using conventional techniques theresulting pellets.

In one embodiment, the core, or pellet, of the invention has an averageweight particle size in the range from 250 to 3000 μm, in particular 500to 3000 μm. Several representative size ranges can be preferred. Forexample, the core size can be comprised between 500 and 1000 μm, orbetween 800 and 1600 μm. In the context of the present invention, theweight average particle size is determined by sieving differentfractions in size, weighting the fractions and calculating the averageparticle size from the weights. The method is well known to a skilledperson in the field of the invention.

It has unexpectedly been found that the mixture of an adsorbent, inparticular activated charcoal, and carrageenan has good formulationproperties, including:

suitable flow characteristics which allows mass transport duringextrusion process,

self lubricating properties with limited sticking to material,

sufficient rigidity to keep the shape of the extrudate,

firmness of the extrudate and enough brittleness which allows smoothcutting of the extrudate, and

minimum plasticity, which allows good spheronization.

None of these advantageous properties has been reported in the priorart.

The invention thus also relates to a composition comprising a mixture ofan adsorbent, preferably activated charcoal, with carrageenan (inparticular kappa-carrageenan). In a further embodiment, said mixture isin the form of a particle (a compact mixture obtainable, for example, byan extrusion spheronization process), also termed a pellet in thepresent application.

Those skilled in the art will recognize that the core composition canfurther include conventional excipients such as antiadherents, binders,fillers, diluents, flavours, colours, lubricants, glidants,preservatives, sorbents and sweeteners. The amounts of such excipientscan vary, but will typically be in the range of 0.1 to 50% by weight ofthe pellet. Of course, the person skilled in the art will adapt theseamounts so that the added excipient does not negatively impact on theadvantageous properties of the mixture of carageenan with the adsorbent.

External Enteric Coating

The core of the formulation can be layered with a coating such that thedrug is released from the formulation in a desired part of theintestine. Several systems are known to those skilled in the art fordelivery of an agent to the different parts of the intestine. Acomprehensive review of the different systems that can be implemented isprovided in Pinto et al., Int J Pharm. 2010 Aug. 16; 395(1-2):44-52.

In a particular embodiment of the invention, the core of the formulationcan be layered with a coating such that the drug is released from theformulation in the lower part of the intestine, i.e in the late ileum,caecum and/or colon. Any coating can be used which ensures that theformulation will not release the adsorbent until it is in the desiredpart of the intestine, namely in the late ileum, the caecum or thecolon. The coating may be selected from coatings which are pH-sensitive,redox-sensitive or sensitive to particular enzymes or bacteria. Entericcoatings are well known to those skilled in the art (for example,reference is made to Chourasia M K and Jain S K, “Pharmaceuticalapproaches to colon targeted drug delivery systems”, J Pharm PharmaceutSci 6(1): 33-66, 2003).

Preferred coating materials are those which are pH sensitive, i.e.pH-dependent enterosoluble polymers. As will be apparent in thefollowing parts of the application, the choice of the pH-dependententerosoluble polymer can be made by taking into account the pH profileof the gastro intestinal tract of the mammal who will be the recipientof the treatment (also herein referred to as the “host being treated”).

The term “enterosoluble polymer” denotes a polymer that is stable anddoes not dissolve in the stomach and the upper parts of thegastrointestinal tract, but readily dissolves when it arrives at thedesired part of the gut to release the active material containedtherein. The solubility of a pH-dependent enterosoluble polymer dependson the conditions of acidity or alkalinity found all along the gut.

In a particular embodiment, the pH-dependent enterosoluble polymer canbe selected among cellulose acetate trimellitate (CAT), celluloseacetate phthalate (CAP) such as Aquateric®, anionic copolymers based onmethylacrylate, methylmethacrylate and methacrylic acid such asEudragit® FS30D, Hydroxypropyl methylcellulose phthalate (HPMCP),Hydroxypropylmethylcellulose acetate succinate (HPMCAS) LF, LG, MF, MGor HF Grades such as Aqoat®, methacrylic acid and ethyl acrylatecopolymers such as Eudragit® L100-55, methacrylic acid and ethylacrylate copolymer such as Eudragit® L30D-55, methacrylic acid andmethyl methacrylate copolymers (1:1 ratio) such as Eudragit® L-100 andEudragit® L12,5, methacrylic acid and methyl methacrylate copolymers(1:2 ratio) such as Eudragit®S-100 and Eudragit® S12,5, Polyvinylacetate phthalate (PVAP) such as Sureteric® and Opadry® and Shellacresins such as SSB® Aquagold.

In a preferred embodiment, the pH-dependent enterosoluble polymer usedin the external layer dissolves at a pH equal to 6.0 and above. Evenmore preferably, it dissolves at a pH equal to 7.0 and above. In thiscontext, the polymer may in particular be selected in the groupconsisting of shellac such as SSB® Aquagold, anionic copolymers based onmethyl acrylate, methyl methacrylate and methacrylic acid such asEudragit® FS30D, methacrylic acid and methyl methacrylate copolymers(1:2 ratio) such as Eudragit®S-100 and Eudragit® S12,5, HPMCAS such asAqoat® AS-MF, MG or HF grades or hydroxypropyl methylcellulose phthalate(HPMCP) such as HP-55 grade.

The above referred to Eudragit® copolymers are commercialized by Evonik.Their composition is known to the skilled artisan and may be found, inparticular, in US 2008/0206350 (U.S. Ser. No. 12/034,943).

The pH dependent enterosoluble polymer is selected first for its abilityto resist acidic pH found into the upper part of the gastro-intestinaltract (GIT) of most mammals and second to fulfill requirement ofdelivering the active agent into the lower part of the intestine, i.e.preferentially the late ileum, the caecum or the colon.

The person skilled in the art knows that in many mammals, the physiologyof the GIT can vary both in terms of pH, length, and transit time. Table1 below represents the various physiological characteristics of somemammals.

TABLE 1 Various intestinal pH found in the gut of different mammalsStomach pH Small intestine pH Species Anter-poster upper-lower Caecum pHColon pH Man 1.7-5.0 5.6--7.5 5.9 5.5/7 Pig 4.3-2.2 6.0-7.5 6.3 6.8 Dog5.5-3.4 6.2-7.5 6.4 6.5 Cat 5.0-4.2 6.2-7.6 6.0 6.2 Horse 5.4-3.36.7-7.9 7.0 7.4 Poultry 4.9-4.2 5.8-7.7 7.0 ND From KararliTT., BiopharmDrug Dispos. 1995 Jul; 16(5): 351-80. Comparison of the gastrointestinalanatomy, physiology, and biochemistry of humans and commonly usedlaboratory animals. Stevens C. E., and Hume, I. D. 1995. ComparativePhysiology of the Vertebrate Digestive System. 2nd ed. New York:Cambridge University Press.

It can be seen from table 1 that most of the enterosoluble polymers willbegin to dissolve in the upper part of the small intestine and, thanksto the thickness of the external coating, the adsorbent will be releasedinto the lower part of the intestine by the time dissolution isachieved.

The coating thickness can be adapted to finely tune the release of theadsorbent into the desired part of the intestine. For example, theenterosoluble polymer layer can represent from 10% to 40% in weight ofthe weight of the total formulation. In a preferred embodiment, theamount of enterosoluble layer is at least 15% of the total weight of theformulation. In a preferred embodiment, the enterosoluble polymer layerrepresents from about 15% to about 35% by weight of the totalformulation, even more preferably, from about 15% to about 20%. In aparticular embodiment, the enterosoluble polymer layer is present in theformulation in an amount of about 15% by weight of the totalformulation.

The type and/or amount of enterosoluble polymer which can be used tocoat the core of the invention may be selected by using the Biodisdissolution tester (USP III release apparatus) as provided in theexamples.

The pH-dependent enterosoluble coating can also include variouscombinations of different pH-dependent enterosoluble polymers. Thoseskilled in the art are able to select such mixtures of pH-dependentpolymers taking into account their general knowledge in this field. Forexample, as mentioned in the above cited article of Chourasia and Jain,a combination of two methacrylic acid polymers such as Eudragit® L100-55and Eudragit® S100 can be provided around the core of the invention.

In a particular embodiment of the invention, the external coatingcontains Eudragit FS30D, or a mixture of Eudragit FS30D and EudragitL30D-55 in a weight ratio comprised in particular between 99:1 to 80:20(FS30D:L30D-55).

In a particular embodiment, the pH-dependent enterosoluble polymer isselected from

shellac,

anionic copolymers based on methyl acrylate, methyl methacrylate andmethacrylic acid,

mixtures of methyl methacrylate and methacrylic acid such as Eudragit®FS30D and methacrylic acid and ethyl acrylate copolymer such asEudragit® L30D-55, in a ratio comprised between 99:1 and 80:20, and

methacrylic acid and methyl methacrylate copolymers (1:2 weight ratio).

In a further particular embodiment, the formulation according theinvention comprises:

a core containing a mixture of activated charcoal with carrageenan(preferably kappa carrageenan), and

a layer of an anionic copolymer based on methyl acrylate, methylmethacrylate and methacrylic acid, such as Eudragit® FS30D.

In a further particular embodiment, the formulation according theinvention comprises:

a core containing a mixture of activated charcoal with carrageenan(preferably kappa carrageenan), and

a layer of a mixture of methyl methacrylate and methacrylic acid such asEudragit® FS30D and methacrylic acid and ethyl acrylate copolymer suchas Eudragit® L30D-55, in a ratio comprised between 99:1 and 80:20.

In another particular embodiment, the formulation according theinvention comprises

a core containing a mixture of activated charcoal with carrageenan(preferably kappa carrageenan), and

a layer of shellac.

The external enterosoluble layer may be applied onto the core by anysuitable means known to a person skilled in the art. For example, it canbe applied using classical fluid bed technology where a water-based orsolvent-based solution of coating is applied by spray-drying onto thecore pellet. When the weight gain is reached, the formulation can bedried and a further coating can be applied. Multiple coatings can thusbe applied successively using spray drying technology.

Furthermore, the colonic region has a high presence of microbialanaerobic organisms providing reducing conditions. Thus the externalcoating may suitably comprise a material which is redox-sensitive. Suchcoatings may comprise azopolymers which can for example consist of arandom copolymer of styrene and hydroxyethyl methacrylate, cross-linkedwith divinylazobenzene synthesized by free radical polymerization, theazopolymer being broken down enzymatically and specifically in thecolon, or disulphide polymers (see PCT/BE91/00006).

Other materials providing release in the colon are amylose, for examplea coating composition can be prepared by mixing amylose-butan-1-olcomplex (glassy amylose) with Ethocel aqueous dispersion (Milojevic etawl., Proc. Int. Symp. Contr. Rel. Bioact. Mater. 20, 288, 1993), or acoating formulation comprising an inner coating of glassy amylose and anouter coating of cellulose or acrylic polymer material (Allwood et al GB9025373.3), pectin, a polysaccharide which is degraded by colonicbacterial enzymes (Ashford et al., Br Pharm. Conference, 1992, Abstract13), reticulated into a gel by divalent cations such as calcium(Rubenstein et al., Pharm. Res., 10, 258, 1993) or zinc (El-Gibaly, Int.J. Pharmaceutics, 232, 199, 2002), chondroitin sulphate (Rubenstein erawl., Pharm. Res. 9, 276, 1992) and resistant starches (Allwood et nl.,PCT WO 89/11269, 1989), dextran hydrogels (Hovgaard and Brondsted, 3rdEur. Symp. Control. Drug Del., Abstract Book, 1994, 87) modified guargum such as borax modified guar gum (Rubenstein and Gliko-Kabir, S.T.P.Pharma Sciences 5, 41-46, 1995), P-cyclodextrin (Siekeer al., Eu. J.Pharm. Biopharm. 40 (suppl), 335, 1994), saccharide containing polymersby which a polymeric construct is included comprising a syntheticoligosaccharide-containing biopolymer including methacrylic polymerscovalently coupled to oligosaccharides such as cellobiose, lactulose,raffinose, and stachyose, or saccharide-containing natural polymersincluding modified mucopolysaccharides such as cross linked chondroitinsulfate; methacrylate-galactomannan (Lehmann and Dreher, Proc. Int.Symp. Control. Rel. Bioact. Mater. 18, 331, 1991) and pH sensitivehydrogels (Kopecek et al., J. Control. Rel. 19, 121, 1992). Resistantstarches, eg glassy amylose, are starches that are not broken down bythe enzymes in the upper gastrointestinal tract but are degraded byenzymes in the colon.

Intermediate Coating

According to a particular embodiment of the invention, the formulationdescribed above comprises at least one further coating provided betweenthe core and the external enteric coating. This further layer(s) (alsoreferred to as “intermediate coating”) is provided to further delay therelease of the adsorbent when necessary. The intermediate coating is inparticular provided to minimize (preferably to totally prevent) theimpact of the adsorbent on the normal absorption process of atherapeutic agent (for example, an antibiotic) by the host organism whensaid therapeutic agent is administered orally along with the formulationaccording to the invention. This embodiment is particularly suited tothe case where the administered therapeutic agent has a delayedabsorption profile, as a consequence of the time necessary to achievemaximum concentration of the agent into the blood (T_(max)).

According to a particular embodiment, the intermediate coating isprovided onto the core of the invention, and a further coating isapplied with a pH-dependent enterosoluble polymer, such as Eudragit™FS30D (as explained above) or a mixture Eudragit® FS30D and Eudragit®L30D-55, in a ratio comprised between 99:1 and 80:20. The pH-dependententerosoluble polymer protects the core from the acidic environmentfound in the upper part of the gastro-intestinal tract. Once thepH-dependent polymer is dissolved, further delayed release of theadsorbent can be obtained due to the intermediate coating.

The intermediate coating can contain pH-dependent or pH-independentpolymers.

Among the pH-dependent polymers that can be used as intermediatecoating, examples include those described above in “externalenterosoluble layer” part, and in particular shellac type polymers suchas SSB® Aquagold, anionic copolymers based on methyl acrylate, methylmethacrylate and methacrylic acid such as Eudragit® FS30D, methacrylicacid and ethyl acrylate copolymer such as Eudragit® L30D-55, HPMCAS suchas Aqoat AS-MF, MG or HF grades or hydroxypropyl methylcellulosephthalate (HPMCP) such as HP-55 grade. In a particular embodiment, theintermediate coating can be a mixture of pH-dependent polymers such asEudragit® FS30D and Eudragit® L30D-55, in a ratio comprised between 99:1and 80:20

pH-independent polymers can be selected among slowly water solublepolymers and water insoluble polymers. Non limiting examples ofpH-independent water soluble polymers include Polyvinylpyrolidone (PVP)and high molecular weight cellulose polymers such ashydroxypropylmethylcellulose (HPMC), hydroxypropyl cellulose (HPC).Further non limiting examples of pH-independent insoluble polymersinclude ethylcellulose polymers and ethyl acrylate methyl methacrylatecopolymer (such as Eudragit® NE30D).

In a particular embodiment of the invention, the intermediate coatingcontains a mixture of polymers. In a first alternative, the mixture ofpolymers comprises polymers of the same type. For example, the mixturecan comprise a pH-dependent polymer with another pH-dependent polymer, apH-independent soluble polymer with another pH-independent solublepolymer, or a pH-independent insoluble polymer with anotherpH-independent insoluble polymer. In another alternative, the mixture ofpolymers comprises polymers of different types. The mixture can comprisea pH-dependent polymer with a pH-independent polymer (either watersoluble or insoluble), a pH-independent soluble polymer with apH-independent insoluble polymer, or a pH-dependent polymer with apH-independent soluble polymer and a pH-independent insoluble polymer.For example, the intermediate coating can comprise the mixture of apH-dependent polymer with a pH-independent polymer, such as a mixture ofEudragit® L30D55 with Eudragit® NE30D (for example, in a weight ratiobetween about 1:9 and about 9:1, in particular between about 2:8 andabout 3:7).

The preferred coating and coating component weight ratio can be readilydetermined by those skilled in the art, for example, by evaluating therelease profile of the dosage form, as provided in the examples (e.g.see Example 8).

For a pharmaceutical agent given by oral route, for example anantibiotic, which has a T_(max) between about 1 and about 2 hours (suchas ciprofloxacin), the core according to the invention can be coatedwith a single pH-dependent polymer, such as an anionic copolymer basedon methylacrylate, methylmethacrylate and methacrylic acid (such asEudragit® FS30D). Release of the adsorbent is achieved in vitro and invivo (in particular in a human subject) after about 4-6 hours, whichlimits the interaction of the adsorbent with the normal absorptionprocess of the antibiotic, or another pharmaceutical agent. The sametype of formulations can be administered after parenteral administrationof the antibiotic, where residual antibiotic is found in thegastrointestinal tract after bile or intestinal membrane excretion. Inthis case, there is no risk of interaction of the adsorbent with theabsorption process of the antibiotic.

In the case where pharmaceutical agents with delayed absorption (T_(max)above 2 hours), and in particular antibiotics such as third generationcephalosporins, are given by oral route concomitantly with the adsorbentmaterial formulated in a delayed delivery system such as those describedabove, it may be preferable to further delay the release of theadsorbent. This can be achieved, for example, by primarily coating thecore with between about 1 and about 3% ethylcellulose (w/w of the totalformulation), preferably 1.5-2.5% (w/w of the total formulation), morepreferably with 2% ethylcellulose or a mixture of Eudragit® L30D-55 withEudragit® NE30D (between 10-40%, preferably between 15-35% w/w of thetotal formulation) further coated with at least 15% (w/w of the totalformulation) of Eudragit® FS30D.

In a particular embodiment, the intermediate coating is selected inorder to achieve a delay of about 20 minutes to about 2 hours in therelease of the adsorbent, as measured by in vitro testing such as with aBioDis dissolution tester (USP III release apparatus). In this system,the dosage form is successively placed into glass tubes filled withapprox 200 mL of dissolution media with a composition yielding pH,buffer capacity and osmolarity corresponding to the different sectionsof the gastrointestinal tract, such as described by Jantratid et al. inPharm. Res. 25 (2008), 1663-1676. This allows a good simulation of invivo release before testing into mammals. pH, fed vs fasted state, andvarious other physiological conditions can be tested. Using the BioDissystem, it is possible for those skilled in the art to finely tune theformulation to achieve a desired pre-determined delayed release.

According to the above, a particular embodiment of the invention relatesto a formulation comprising:

a core comprising a mixture of an adsorbent with carrageenan,

an external layer of a pH-dependent enterosoluble polymer, and

a intermediate coating provided between the core and the external layer.

In a particular embodiment, the invention relates to a formulationcomprising:

a core comprising a mixture of activated charcoal with carrageenan(preferably kappa-carrageenan),

a intermediate coating selected in the group consisting of HPMC,ethylcellulose and a mixture of methacrylic acid and ethyl acrylatecopolymer such as Eudragit® L30D-55 and ethyl acrylate methylmethacrylate copolymer such as Eudragit® NE30D (for example in a mixtureratio of 1:9 to 9:1, preferably of 2:8 to 3:7), and

an external layer of an anionic copolymer based on methyl acrylate,methyl methacrylate and methacrylic acid, such as Eudragit® FS30D.

In another particular embodiment, the formulation of the inventioncomprises:

a core comprising a mixture of activated charcoal with carrageenan(preferably kappa-carrageenan),

a 1-3% ethylcellulose intermediate coating, preferably a 1.5-2.5%ethylcellulose coating, most preferably a 2% ethylcellulose intermediatecoating (w/w of the total formulation), and

a 15% (w/w of the total formulation) external layer of an anioniccopolymer based on methyl acrylate, methyl methacrylate and methacrylicacid, such as Eudragit® FS30D.

In a further particular embodiment, the formulation of the inventioncomprises:

a core comprising a mixture of activated charcoal with carrageenan(preferably kappa-carrageenan),

a 15-35% (w/w of the total formulation) intermediate coating made of a2:8 to 3:7 mixture of methacrylic acid and ethyl acrylate copolymer(such as Eudragit® L30D-55) and ethyl acrylate methyl methacrylatecopolymer (such as Eudragit® NE30D), and

a 15% (w/w of the total formulation) external layer of an anioniccopolymer based on methyl acrylate, methyl methacrylate and methacrylicacid, such as Eudragit® FS30D.

In another particular embodiment, the formulation of the inventioncomprises:

a core comprising a mixture of activated charcoal with carrageenan(preferably kappa-carrageenan),

a 1-3% ethylcellulose intermediate coating, preferably a 1.5-2.5%ethylcellulose coating, most preferably a 2% ethylcellulose intermediatecoating (w/w of the total formulation), and

a 15% to 35% (w/w of the total formulation) external layer of a mixtureof methyl methacrylate and methacrylic acid such as Eudragit® FS30D andmethacrylic acid and ethyl acrylate copolymer such as Eudragit® L30D-55,in a ratio comprised between 99:1 and 80:20.

In a further particular embodiment, the formulation of the inventioncomprises:

a core comprising a mixture of activated charcoal with carrageenan(preferably kappa-carrageenan),

a 15-35% (w/w of the total formulation) intermediate coating made of a2:8 to 3:7 mixture of methacrylic acid and ethyl acrylate copolymer(such as Eudragit® L30D-55) and ethyl acrylate methyl methacrylatecopolymer (such as Eudragit® NE30D), and

a 15% to 35% (w/w of the total formulation) external layer of a mixtureof methyl methacrylate and methacrylic acid such as Eudragit® FS30D andmethacrylic acid and ethyl acrylate copolymer such as Eudragit® L30D-55,in a ratio comprised between 99:1 and 80:20.

Dosage Forms

In another aspect, the present invention provides pharmaceuticallyacceptable dosage forms which comprise a therapeutically-effectiveamount of one or more of the adsorbents described above, formulatedtogether with carrageenan and one or more pharmaceutically acceptableadditives. As described in detail below, the dosage forms of theinvention can be specially formulated for administration in solid form.

The phrase “therapeutically-effective amount” as used herein means thatamount of one or more of the compounds described above, material, orformulation comprising one or more of the compounds described abovewhich is effective for producing some desired therapeutic effect.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, formulations, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable additive” as used herein means apharmaceutically-acceptable material, formulation or vehicle, such as asolid filler, diluent, excipient involved in carrying or transportingthe subject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each additive must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient.

Dosage forms that contain multiple units, such as pellets individuallycoated by enterosoluble polymers such as the one described above, can bepreferred in order to improve the in vivo dispersion of the activatedcharcoal. Such pellets present more practical flexibility, becausecoating can be directly achieved on their surface, for example, using afluid bed system.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the dosage form.

Dosage forms of the present invention include those suitable for oraladministration. The formulations can conveniently be presented in unitdosage form and can be prepared by any methods well known in the art ofpharmacy.

Dosage forms of the invention suitable for oral administration can be inthe form of capsules, tablets, sachets, each containing a predeterminedamount of the adsorbent formulation.

A tablet can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate, cross-linked sodium carboxymethyl cellulose orpolysaccharide), surface-activated or dispersing agent. Molded tabletscan be made by molding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent such as water.

The solid dosage forms described above can be combined in a final dosageform comprising single or multiple units. Examples of multiple unitsinclude multilayer tablets, capsules containing tablets, pellets,granules, etc.

The core of the invention can be coated with an external layer, andoptionally an intermediate coating as provided above. The coatedformulation (coated with an external enteric coating, and comprising ornot an intermediate coating) or uncoated core can further be combined ina unit drug dosage form, such as a tablet, capsule, and the like, whichcan be further coated with a coating material for effectivedelayed-release which include, but are not limited to, cellulosicpolymers such as hydroxypropyl cellulose, hydroxyethyl cellulose,hydroxymethyl cellulose, hydroxypropyl methyl cellulose,methylcellulose, carboxymethylcellulose sodium, copolymers such aspolyvinyl pyrrolidone; hydroxypropyl methyl cellulose acetate succinate,hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate,cellulose acetate trimellitate and acrylic acid polymers and copolymers,preferably formed from acrylic acid, methacrylic acid, methyl acrylate,ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and othermethacrylic resins that are commercially available under the tradenameEudragit®. (Rohm Pharma; Westerstadt, Germany), including Eudragit®L30D-55 and L100-55 (soluble at pH 5.5 and above), Eudragit® L-100(soluble at pH 6.0 and above), Eudragit® S (soluble at pH 7.0 and above,as a result of a higher degree of esterification), and Eudragit FS30D aanionic copolymer of methacrylic acid, methyl acrylate andmethylmethacrylate; ethyl cellulose, cellulose acetate; Eudragit® NE, RLand RS (water-insoluble polymers having different degrees ofpermeability and expandability) vinyl acetate, vinylacetate phthalate,vinylacetatecrotonic acid copolymer, and ethylene-vinyl acetatecopolymer; vinyl polymers and; Enzymatically degradable polymers such asazo polymers, pectin, chitosan, amylose and guar gum; zein and shellac.

The preferred coating weights for particular coating materials can bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, pellets and granules prepared withdifferent quantities of various coating materials.

It is the combination of materials, method and form of application thatproduce the desired release characteristics.

The coating formulation can include conventional additives, such asplasticizers, pigments, colorants, stabilizing agents, glidants, etc. Aplasticizer is normally present to reduce the fragility of the coating,and will generally represent about 5 wt. % to 50 wt. % relative to thedry weight of the polymer. Examples of typical plasticizers includepolyethylene glycol, propylene glycol, triacetin, dimethyl phthalate,diethyl phthalate, dibutyl phthalate, dibutylsebacate, triethyl citrate,tributyl citrate, triethyl acetyl citrate, castor oil and acetylatedmonoglycerides. A stabilizing agent is preferably used to stabilizeparticles in the dispersion. Typical stabilizing agents are nonionicemulsifiers such as sorbitan esters, polysorbates andpolyvinylpyrrolidone. Glidants are recommended to reduce stickingeffects during film formation and drying, and will generally representapproximately 0 wt. % to 100 wt. % of the polymer weight in the coatingsolution. One effective glidant is talc. Other glidants such asmagnesium stearate and glycerol monostearates can also be used. Pigmentssuch as titanium dioxide can also be used. Small quantities of ananti-foaming agent, such as a silicone (e.g., simethicone), can also beadded to the coating formulation.

These dosage forms can be administered to humans and animals for therapyby any suitable route of administration.

Actual dosage levels of the adsorbent in the dosage form of thisinvention can be varied so as to obtain an effective removal of anyresidual antibiotic or other pharmaceutical agents or toxin in theintestinal tract, for a particular patient, formulation, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalformulation required. For example, the physician or veterinarian couldstart with doses of the compounds of the invention employed in thepharmaceutical formulation at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above.

If desired, the effective daily dose of the active compound can beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

As already mentioned, the formulation according to the invention may beused in a method for eliminating the adverse effects of therapeuticagents, in particular, but not only, of antibiotics. According to aparticular embodiment of this method, the formulation of the inventionand the therapeutic agent are administered simultaneously. As such theamount of adsorbent may be adapted to the amount of therapeutic agentadministered to the subject in need thereof. In this case, the weightratio between the adsorbent and the antibiotic agent may be above 1,more preferably above 2, even more preferably above 3, and mostpreferably above 9.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The subject receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; poultry and pets in general may also berecipients of such a treatment.

The administration of the formulation according to the invention to ananimal is preferably carried out by including it in the animal's food.This is preferably accomplished by preparing an appropriate feed premixcontaining the formulations according to the invention in an effectiveamount and incorporating the premix into the complete ration.Accordingly, the present invention also relates to an animal food premixcomprising food and formulations as described above. The invention alsorelates to an animal food ration comprising the formulations accordingto the invention.

Applications

Therapeutic Applications:

The formulations according to the invention can be used to treatconditions and disorders for which intestinal delivery of adsorbents isappropriate. Accordingly, the invention also relates to a formulation asdescribed above, for use as a medicament.

The formulation according to the invention can be used to adsorb andtherefore remove from the intestine any drug, metabolite or prodrugthereof, or toxin. This may be done after oral or parenteraladministration of an active drug, which could be useful for limiting ordecreasing adverse effects in the subject being treated when they reachthe lower intestine and/or colon.

As such, the present invention relates to the formulation as describedabove, for use in a method for eliminating drugs in the intestinal tractbefore they reach the colon or as they reach the colon, preferablybefore they reach the caecum or as they reach the caecum and proximalcolon.

The invention further provides a method for eliminating drugs in theintestinal tract before they reach the colon or as they reach the colon,preferably before they reach the caecum or as they reach the caecum andproximal colon, comprising administering to a patient in need thereof aformulation according to the invention.

Furthermore, the invention provides a formulation as described above,for use in a method for reducing or eliminating the side effect(s) of adrug in the intestinal tract, wherein the formulation eliminates thedrug before it reaches the colon or as it reaches the colon, preferablybefore it reaches the caecum or as it reaches the caecum and proximalcolon.

The terms “drug”, “therapeutic agent” and “pharmaceutical agent”, andterms derived therefrom, are herein used interchangeably and refer to acompound that provides a desired biological or pharmacological effectwhen administered to a human or animal.

Conditions and disorders that may be treated with the formulationaccording to the invention may be those that result from exposure of thecolon to antibiotics, such as the development of antibiotic resistance,antibiotic treatment-associated development of C. difficile (or otherpathogenic bacteria), antibiotic treatment-associated fungal infectionsor antibiotic treatment-associated diarrhea. The adsorbent will adsorbresidual antibiotics, and the formulations according to the inventioncan be administered in a therapeutically effective dosage to a patientwho has been, is being, or will be administered an antibiotic. Anyantibiotic that can be adsorbed into/onto the adsorbent can beinactivated and has no antibiotic activity once fully adsorbed.Representative examples of antibiotic classes that can be adsorbedinclude beta-lactams, cyclines, macrolides, quinolones, aminoglycosides,glycopeptides, sulfonamides, phenicols, furans, polypeptides,oxazolidones and antibiotics such as fosfomycin, rifampin and the like.

The invention thus also relates to a formulation as described above, foruse in a method for eliminating residual antibiotics in the intestinaltract, preferably before they reach the colon or as they reach thecolon. More preferably, the formulation is used in a method foreliminating residual antibiotics in the intestinal tract, preferablybefore they reach the caecum or as they reach the caecum and proximalcolon. According to the invention, the adsorbent is preferably deliveredbetween the part of the intestine where the antibiotics are absorbed(duodenum and jejunum) and where their deleterious effect on thecommensal bacteria occur (caecum and colon). The invention furtherrelates to a method for eliminating residual antibiotics in theintestinal tract, preferably before they reach the colon or as theyreach the colon, most preferably before they reach the caecum or as theyreach the caecum and proximal colon comprising administering to asubject in need thereof an effective amount of the formulation of theinvention.

The invention further relates to a formulation as described above, foruse in a method for eliminating the adverse effects of antibiotic agentsin the intestinal tract, in particular for eliminating the developmentof antibiotic resistance, antibiotic treatment-associated development ofC. difficile (or other pathogenic bacteria), antibiotictreatment-associated fungal infections or antibiotictreatment-associated diarrhea. The invention further relates to a methodfor eliminating the adverse effects of antibiotic agents in theintestinal tract, comprising administering to a subject in need thereofan effective amount of the formulation of the invention.

In another embodiment, the formulation of the invention is administeredto a patient who suffers from a disorder treated with pharmaceuticalagents which have side effects when they reach the lower part of theintestine, in particular when they reach the colon. As developed below,Irinotecan is a representative compound having such behavior.

In particular embodiments, the formulation is administered to a patientwho suffers from a disorder treated with pharmaceutical agents whichbind to relevant receptors in the body of the patient other than in thecolon to treat the disorder, but which, when bound to receptors in thecolon, result in side effects. For example, the colon includescholinergic and serotonin receptors, which are also present in thecentral nervous system. Treatment with agents that bind to cholinergicreceptors can result in side effects if the compounds bind to receptorsin the colon. Co-administration of the formulation of the invention withthe agents that bind to such receptors can minimize or eliminate theseside effects.

The invention thus also relates to a formulation as described above, foruse in a method for eliminating the side effects in the intestine, inparticular in the colon, of pharmaceutical agents administered as atreatment for a disorder, but that have side effects when they reach thelate ileum, the caecum or the colon. The present invention can alleviateor eliminate these side effects. The invention further relates to amethod for eliminating the side effects in the intestine, in particularin the colon, of pharmaceutical agents, in particular pharmaceuticalagents administered as a treatment for a disorder, but that have sideeffects when they reach the late ileum, the caecum or the colon,comprising administering to a subject in need thereof an effectiveamount of the formulation of the invention. In particular, the presentinvention can be used to alleviate or eliminate an inflammation and/ordiarrhea induced by a treatment with a pharmaceutical agent.

Irinotecan is an illustrative, non-limiting, example of a pharmaceuticalagent administered as a treatment for a disorder, but that has sideeffects when it, and/or its metabolites, reach the late ileum, thecaecum or the colon. A particular embodiment of the invention provides aformulation for eliminating or reducing Irinotecan-induced diarrhea, inparticular Irinotecan-induced late-onset diarrhea.

Irinotecan (also known as CPT-11), a semi-synthetic analogue of thenatural alkaloid camptothecin, is a soluble prodrug of7-ethyl-10-hydroxycamptothecin (SN-38) which is a topoisomerase Iinhibitor which has 1000-fold more potent antineoplastic activity thanthe prodrug form in vitro. Irinotecan has more particularly beenapproved by the FDA for metastatic colorectal cancer in 1998. It ismostly used as first line in combination regimens or as a single agentafter failure of 5-fluorouracil (5-FU)-based therapy. However, it hasbeen found that late-onset diarrhea is a major dose-limiting toxicity ofIrinotecan. Accumulation of SN-38 in the intestine is the main cause ofIrinotecan-induced late stage diarrhea.

More generally, diarrhea often develops as a side effect during clinicaltreatment with chemotherapeutic agents. This adverse effect is mostcommonly associated with chemotherapeutic agents such as 5-fluorouracil,cisplatin or Irinotecan. In particular, late-onset diarrhea due to theadministration of Irinotecan can be persistent, may lead to dehydrationand electrolyte imbalance and can be, in some cases, sufficientlyserious (grade 3 or 4 diarrhea) that Irinotecan administration must bemodified, interrupted or discontinued. Diarrhea constitutes aproblematic symptom for patients, and because it may provoke reductionsin Irinotecan doses or the frequency of Irinotecan administration,diarrhea may compromise the therapeutic efficiency of Irinotecan whichis highly dependent on the administered dose.

A sign of the importance and frequency of this side effect is the factthat a protocol for treatment by loperamide, in case diarrhea occurs, iseven indicated on the labeling of Irinotecan. Indeed, in humans, theintensive and immediate administration of loperamide (an agent thatslows intestinal motility and affects water and electrolyte movementthrough the bowel) is used to reduce or control diarrhea once diarrheahas started. However, loperamide has side effects on its own, such asinducing intestinal occlusion (Hanauer, S B, Rev Gastroenterol Disord. 8(2008), 15-20).

The prevention of Irinotecan-induced diarrhea with activated charcoalhas been previously proposed (Michael et al., Journal of ClinicalOncology, Vol. 22, No. 21, Nov. 1, 2004). However, the treatmentconsisted in the oral administration of non-formulated activatedcharcoal. This raises at least two issues, both related to the nonspecific nature of this adsorbent. One of these issues is the likelysaturation of activated charcoal by digestive material as it progressesthrough the gastrointestinal tract. It would be preferable to provide tothe terminal parts of the intestine a maximally active adsorbent inorder to obtain a strong adsorption of Irinotecan and/or its metabolitesat the place where they elicit their unwanted effects. The secondproblem is related to the fact that Irinotecan is often administeredwithin a multi-drug treatment regimen, which may comprise drugsadministered orally. In particular, Irinotecan can be administered inassociation with 5-fluorouracil and leucovorin; other drugs may beadded, as required for various reasons, to the treatment.Co-administration of non-formulated charcoal in this context isundesirable since the adsorbent may adsorb the co-administered drug(s)and thus prevent them to elicit the desired effects they have been usedfor.

The present invention is advantageous in that it allows eliminating orreducing the adverse effects of Irinotecan, in particularIrinotecan-induced diarrhea (most particularly Irinotecan-inducedlate-onset diarrhea) without eliciting further adverse effects ortoxicity. Furthermore, thanks to the present invention, Irinotecan canbe used at its most effective therapeutic dose since no alteration ofthe dosage regimen is necessary because of the elimination of theadverse effects of Irinotecan. In preventing diarrhea symptoms inpatients receiving Irinotecan therapy, the formulation of the presentinvention has the potential to reduce the incidence, severity, and/orduration of diarrhea, improve patient quality of life, avoiddiarrhea-related hospitalization, and/or prevent Irinotecan dosereduction, treatment interruption, or discontinuation.

The method of the invention also provides elimination or reduction ofmetabolites of Irinotecan, in particular SN-38, and the elimination orreduction of the adverse effect of such Irinotecan metabolites.

The person skilled in the art will recognize that these advantages arealso provided for therapy with molecules other than Irinotecan, thatelicit adverse effects, in particular molecules that induce diarrhea,when they reach the lower part of the gastrointestinal tract. Suchmolecules could be other analogues and derivatives of camptothecin, suchas Topotecan, and other drugs used in cancer chemotherapy.

Colchicine, a drug used for pain and gout arthritis treatment is anotherrepresentative example of a pharmaceutical agent whose eliminationaccording to the invention would be advantageous.

It is also known that gastrointestinal problems are commonly reportedbecause of adverse drug reactions with blood pressure medications(Calcium Channel blockers), pain medications (especially narcotics),antidepressants, antacids that contain aluminum and calcium,antiparkinson drugs, antispasmodics, diuretics, and anticonvulsants, andthat many drug classes are associated with constipation. Often,constipation persists, and patients discontinue treatment because theside effect is burdensome. Drugs such as risperidone can be associatedwith colonic disorders, such as megacolon (Lim et al, Singapore Med J2002, Vol 43(10): 530-532). The formulation of the invention can beadministered to a patient in need thereof to treat these problems.

Thus, in a particular embodiment, the invention relates to a formulationas described above, for use in a method for eliminating the side effectsin the intestine, in particular in the colon, of a therapeutic agent,for example of a chemotherapeutic agent, in particular of Irinotecan andderivatives thereof (in particular its metabolite SN-38). The inventionfurther relates to a method for eliminating the side effects in theintestine, in particular in the colon, of a therapeutic agent, forexample of a chemotherapeutic agent, in particular of Irinotecan andderivatives thereof (in particular its metabolite SN-38) when it reachesthe late ileum, the caecum or the colon, comprising administering to asubject in need thereof an effective amount of the formulation of theinvention.

The invention further relates to a method for treating cancer (inparticular metastatic colorectal cancer) with a chemotherapeutic agent,in particular with Irinotecan, comprising administering to a patient inneed thereof

-   -   an effective amount of the chemotherapeutic agent, and    -   an effective amount of the formulation according to the        invention.

The invention also relates to a method for reducing or eliminating theneed to decrease the dose, interrupt or discontinue the use of atherapeutic agent, for example of a chemotherapeutic agent, inparticular Irinotecan, comprising administering a formulation accordingto the invention to a patient in need of a therapy by said therapeuticagent.

The formulation according to the invention may be administered before,with or after administration of the therapeutic agent which is intendedto be eliminated from the lower parts of the gastroinstestinal tractaccording to the invention. Preferably, the formulation according to theinvention is administered before, or together with the therapeuticagent. For example, the subject takes at the same time an antibiotic (oranother therapeutic agent, for example a chemotherapeutic agent likeIrinotecan, etc.) and a formulation according to the invention.

Thus, for example, administration of the therapeutic agent and of theformulation according to the invention can be simultaneous or sequential(the formulation according to the invention being administered before orafter administration of the therapeutic agent), as a single dose orrepeated several times a day, for one day or several days.Administration of the formulation of the present invention can beginbefore administration of the therapeutic agent, and continued after saidadministration of the therapeutic agent.

Furthermore, the formulation of the invention can also be administeredbefore or after, preferably before, the onset of the adverse effect tobe eliminated. In an illustrative embodiment, the formulation of theinvention is administered before the patient is treated with adiarrhea-inducing therapeutic agent like Irinotecan, colchicine, orothers. The formulation of the invention can be administered once or atmultiple times, for example every four or six hours one or two daysprior to, as well as after administration of the therapeutic agent,during one or several days.

In a particularly preferred embodiment, in the context of a treatment ofa patient with Irinotecan, the formulation of the invention isadministered before the administration of Irinotecan to the patient, forexample one or two days before, once or several times a day (for exampleat each meal), and administration of the formulation is continued on theday of administration of Irinotecan and at least 4 days afteradministration of Irinotecan, preferably several times a day. Ideally,the treatment is continued between 4 to 10 days, preferably 7 days afteradministration of Irinotecan to make sure that all remaining residualtraces of Irinotecan or its metabolites are eliminated from theintestine of the patient.

The invention also relates to a kit comprising at least a firstformulation comprising a therapeutic agent whose presence is unwanted inthe lower parts of the intestine, and a formulation containing anadsorbent as described above. The invention further relates to a kitaccording to the invention, for use in one of the methods describedabove, comprising the administration of the formulations of the kit to asubject in need thereof. The formulations are administrated sequentially(one before the other) or simultaneously, preferably simultaneously.

The invention further relates to a method for the treatment of a diseasestate in a subject in need thereof, comprising:

administering to the subject a pharmaceutical agent useful for thetreatment of the disease, in particular an antibiotic (or any otherpharmaceutical agent having side effects when it reaches the lower partof the intestine, as described above), and

administering to the same subject, either sequentially (before or afteradministration of the pharmaceutical agent) or simultaneously theformulation according to the invention, for eliminating or reducing theamount of the pharmaceutical agent in the lower part of the intestine(i.e., the late ileum, the caecum or the colon).

Representative, non limiting, examples of pharmaceutical agents that canbe used in the treatment of a disease state along with the formulationof the invention include antineoplastic agents, for exampletopoisomerase I inhibitors such as camptothecin derivatives likeIrinotecan or Topotecan, anti-inflammatory compounds or inhibitors ofinterleukin-1 such as diacerhein, pancrelipase (such as Pancrease,Creon, Zenpep), selective phosphodiesterase-4 inhibitors used for thetreatment of Chronic obstructive Pulmonary Disease (COPD) such asroflumilast or cilomilast, and compounds having anti-mitotic activitiessuch as colchicine.

As described above, the content of the formulation according to theinvention may be adapted to the absorption profile of most types oftherapeutic agents, and in particular to most kind of antibiotic agents.As an effect, the release of the adsorbent is most reliable andconsistent to achieve no interaction with the normal therapeutic agentabsorption process. Accordingly, and as provided above, the delivery ofthe adsorbent may be delayed in such a way so as to provide delivery ata predetermined time after the therapeutic agent, for example anantibiotic, is completely absorbed to have its therapeutic effect. Thisis achieved through specific coatings, providing both protection in theupper part of the intestinal tract and efficient adsorbentdelayed-release. This provides a major and very innovative advantageover the above-mentioned general and specific approaches.

The sequence of administration can also be adapted by the person skilledin the art. For example, a pharmaceutical treatment may comprise theadministration of the pharmaceutical agent by routes different from theoral route. For example, a pharmaceutical agent can be administered viaa parenteral route, such as by an injection (for example intravenous,intra-arterial, intrathecal, intramuscular injection). In this case, theperson skilled in the art will adapt the timing of administration of theformulation of the invention according to its knowledge of the timing ofexcretion of the pharmaceutical agent in the gastrointestinal tract.

The formulation may also be administered to a patient who suffers fromthe effects of bacterial or fungal toxins on the colon. Examples of suchtoxins include mycotoxins, endotoxins or enterotoxins, such as thoseproduced by Clostridium difficile (believed to be a major cause ofpost-antibiotic diarrhea throughout the world). In this embodiment, theadsorbents are administered in a therapeutically effective dosage toadsorb the toxins.

The invention thus also relates to a formulation as described above, foruse in a method for eliminating the effects of bacterial or fungaltoxins in the colon. The invention further relates to a method foreliminating the effects of bacterial or fungal toxins on the colon,comprising administering to a subject in need thereof an effectiveamount of the formulation of the invention.

Furthermore, the invention also relates to a formulation as describedabove, for use in a method for the treatment of disease statescharacterized by the accumulation of substances in the lower part of thegastroinstestinal tract, this accumulation being responsible for thedevelopment of a number of pathological conditions. For example, theformulation can be useful for the treatment of conditions such as, butnot limited to, hepatic encephalopathy, irritable bowel syndrome,chronic renal disease, C. difficile associated diarrhea or antibioticassociated diarrhea. Representative substances which can be adsorbed bythe formulation disclosed herein include, but are not limited to,ammonia, indoles, advanced glycation end products (AGEs) and certainbacterial toxins.

The formulation of the invention can be administered to a patient whosuffers from Chronic Kidney Disease (CKD). Advanced glycation endproducts (AGEs), phenols (for example p-cresylsulphate) and indoles (forexample, indoxyl sulfate) are representative toxins generated orintroduced in the body via the intestine which can be involved in CKD.Accordingly, in a particular embodiment, the invention relates to theformulation as defined above for use in a method for the treatment ofCKD. The invention more specifically relates to a formulation asdescribed above, for use in a method for eliminating toxins involved inthe generation of uremic retention solutes. The invention furtherrelates to a method for eliminating the effects of toxins involved inthe generation of uremic retention solutes, comprising administering toa subject in need thereof an effective amount of the formulation of theinvention. More specifically, the invention relates to the eliminationor reduction of the amount of AGEs, phenols (for examplep-cresylsulphate) and/or indoles (for example, indoxyl sulfate) in thelower part of the intestine (i.e., the late ileum, the caecum or thecolon).

The formulation of the invention can further be administered to apatient who suffers from Inflammatory Bowel Disease (IBD), in particularfrom ulcerative colitis or Crohn's disease. Thanks to the formulation ofthe invention, it is now possible to induce or re-establishimmunological tolerance by recomposing the commensal microflora in theintestine by adsorbing excess non specific mucosal bacteria oraggressive metabolites and mediators that accumulate in the intestinalmucosa such as nitric oxide, oxygen radicals, prostaglandins,leukotrienes, histamine, proteases, and matrix metallo-proteinases. Theinvention thus relates to the formulation as described above, for use ina method for inducing or re-establishing immunological tolerance in apatient who suffers from an IBD, in particular from ulcerative colitisor Crohn's disease. The invention therefore also relates to a method forthe treatment of an IBD, in particular of ulcerative colitis or Crohn'sdisease, comprising administering to a patient in need thereof aformulation according to the invention. The invention further relates toa formulation as described above for use in a method for eliminating orreducing the amount of excess non specific mucosal bacteria oraggressive metabolites and mediators that accumulate in the intestinalmucosa such as nitric oxide, oxygen radicals, prostaglandins,leukotrienes, histamine, proteases or matrix metallo-proteinases.

The formulation according to the invention can also be used to treatHepatic Encephalopathy (HE). A key role is thought to be played in thisdisorder by circulating gut-derived toxins of nitrogenous compounds,notably ammonia. The formulation according to invention can for examplebe used to adsorb ammonia produced by bacteria in the gut of a patientin need thereof. As such, the invention relates to a formulation asdescribed above, for the elimination or reduction of nitrogenouscompounds, notably ammonia, in the gut of a subject in need thereof. Theinvention also relates to a method for eliminating or reducing theamount of nitrogenous compounds, notably ammonia, in the gut of asubject in need thereof, comprising administering to said patient atherapeutically effective amount of a formulation as described above.

When the subject to be treated is an animal, for example pet or farmanimal, the formulation according to the invention may be incorporatedin food. For example, the formulation according to the invention may beincorporated in a medical food (or drug food) either without or with anantibiotic, if the food is intended to be used as a therapeuticformulation. Alternatively, the formulation according to the inventionmay be in the form of a premix food, which will serve as a foodadditive.

Veterinary Applications:

The formulation according to the invention is able to release anadsorbent in a specific part of the intestine of a subject. As mentionedabove, the subject may be a pet or farm animal. For example, the subjectmay be a pig, a dog, a cat, a horse or fowl.

Adsorbents, besides being useful in a therapeutic context, are able toeliminate a wide range of molecules. Accordingly, the formulationsaccording to the invention may be implemented in methods in which therelease of an adsorbent in the lower parts of the intestine would beadvantageous.

For example, the formulation according to the invention may be used forreducing flatulencies (for example via H₂S adsorption), stool smell (forexample via ammonium adsorption), halitosis, food intolerance, etc.

The present invention will be further understood with reference to thefollowing non-limiting examples.

EXAMPLES Example 1: Kinetics of Levofloxacin Adsorption by ActivatedCharcoal in Simulated Colonic Fluid

A solution of levofloxacin (50 μg/ml) was incubated with non formulatedactivated charcoal (NFAC) in simulated colonic fluid (50 mM sodiumphosphate buffer pH 6.0, 100 mM NaCl) with gentle mixing at 37° C. Theratio of NFAC to levofloxacin was either 3:1 or 10:1.

Samples were withdrawn after 0, 0.5, 1 and 2 h incubation, centrifugedand filtered, and the amount of levofloxacin remaining in thesupernatant was measured by its absorbance at 287 nm. As shown in FIG.1, even with the lowest ratio of NFAC to levofloxacin, all of theantibiotic was adsorbed onto the charcoal after 30 min incubation.

Example 2: Microbiological Assay of Ciprofloxacin and Levofloxacin

The microbiological assay consists in measuring the biological activityof an antibiotic, i.e. its capacity to inhibit the growth of anindicator bacterial strain. To this end, agar plates were made withDifco medium 5, containing E. coli strain CIP 7624 as indicator strain.20 μl samples containing the antibiotic to be measured were spotted ontopaper discs applied directly onto the surface of the agar plates. After18 h incubation at 37° C., the diameters of the zones around the paperdiscs where bacterial growth had been inhibited by the presence of theantibiotic were measured.

As shown in FIG. 2, there is a linear relationship between the logarithmof the concentration of the antibiotic solution (log₁₀ μg/ml) and thediameter (mm) of growth inhibition. The assay was linear from 0.04 to 5μg/ml ciprofloxacin.

When 20 μl of a suspension of non formulated charcoal was spotted ontothe discs, no growth inhibition was observed, showing that charcoalalone did not have any effect on bacterial growth in this assay.

A similar assay was set up for levofloxacin using the same medium andindicator strain; this assay gave a linear response from 0.15 to 10μg/ml levofloxacin (not shown).

Example 3: Kinetics of Ciprofloxacin Adsorption by Activated CharcoalMeasured by Microbiological Assay

A solution of ciprofloxacin (50 μg/ml) was incubated with 150 μg/mlactivated charcoal in modified simulated colonic fluid (18.7 mM maleicacid, 84 mM NaCl, pH 6.0). Samples were withdrawn at various times,centrifugated, and the amount of ciprofloxacin remaining in thesupernatant was measured by a microbiological assay as described inexample 2. As shown in FIG. 3, the result was essentially the same as inthe experiment described in example 1, where antibiotic concentrationswere measured spectrophotometrically. Almost all of the antibiotic wasadsorbed onto the charcoal within one hour. It is worthy to note thatthe sample marked as withdrawn at time zero in fact representedapproximately one minute of contact between ciprofloxacin and thecharcoal; within this short period of time, the charcoal had alreadyadsorbed close to 70% of the antibiotic.

Example 4: Kinetics of Levofloxacin Adsorption by Activated Charcoal inCaecal Medium

In order to mimic the conditions under which activated charcoal wouldinteract with antibiotics in vivo, we measured the adsorption oflevofloxacin on activated charcoal in the presence of intestinal mediumcollected from the caecum of healthy piglets (ex vivo conditions).

Levofloxacin (800 μg/ml) was preincubated with an equal volume of pigletcaecal medium for 2 h at 37° C. with gentle agitation. Similarly, asuspension of non formulated activated charcoal, or deformulated product(deformulated coated pellets, or DCP) containing 80 mg/ml of equivalentactivated charcoal was incubated under the same conditions as above withan equal volume of piglet caecal medium. Deformulation is carried out asprovided in Example 6 below. The antibiotic and charcoal suspensions incaecal medium were then mixed in equal volumes, and incubated for up to5 h at 37° C. under gentle agitation; this represented a 100:1 ratio ofcharcoal to levofloxacin. At the indicated times, samples werewithdrawn, centrifuged, and the amount of free and active antibioticremaining in the supernatant was measured by the microbiological assaydescribed above. FIG. 4 shows that approximately half of the antibioticwas adsorbed onto the caecal medium, reaching equilibrium by one hour.In the presence of activated charcoal, no free and active antibioticremained in the supernatant after one hour, showing that even in thepresence of high amounts of actual intestinal medium, activated charcoalwas able to efficiently adsorb levofloxacin. The experiment furthershows that formulation of the activated charcoal did not affect itscapacity to adsorb levofloxacin under such ex vivo conditions.

The experiment was performed with caecal medium extracted from twodifferent piglets; the mean±SD for triplicate determinations is shown.

Example 5: Desorption of Levofloxacin in Different Conditions

Levofloxacin (200 μg/ml final concentration) was adsorbed onto nonformulated activated charcoal (NFAC) or deformulated coated pellets(DCP) in the presence of piglet caecal medium as described in example 4,except that the ratio of activated charcoal to levofloxacin was 50:1 inthese experiments. After 2 h incubation of Levofloxacin with the ceacalmedium and charcoal, the medium was centrifuged, the pellet containingcharcoal and caecal medium particles was washed 3 times, and finallyincubated for up to 30 days in 50 mM sodium phosphate buffer, containing100 mM NaCl, at pH 4.0, 7.0 or 10.0 with gentle agitation at 22° C. Acontrol was performed with caecal medium, but in the absence ofcharcoal; the dissociation experiments were performed in the same volumeas the original incubation. At indicated times, a sample was withdrawn,centrifuged, and the amount of free and active antibiotic released inthe medium was measured from the supernatant by a microbiological assay.

FIG. 5 shows that some antibiotic was released from material in thecaecal medium over time, and that this amount was much more important atpH 10.0 than at pH 4.0 and 7.0. A lower amount of levofloxacin wasreleased in the presence of NFAC. Quite remarkably, at pH 4.0 and 7.0,the amount of released of charcoal in the presence of DCP was below thedetection limit (0.15 μg/ml levofloxacin). At pH 10.0, the release oflevofloxacin was measurable, but did not exceed 2 μg/ml, representing 1%of the original amount of antibiotic in the experiment. Hence, at pHvalues likely to be encountered in natural media, levofloxacindissociation from activated charcoal contained in DCP could not bemeasured.

Example 6: Adsorption Efficacy of Other Antibiotics onto ActivatedCharcoal

The assay conditions were as follows:

-   -   experiments were performed in phosphate buffer saline (PBS) at        pH 6 adjusted to colon osmolarity,    -   an initial amount of antibiotic of 200 μg/ml was tested (or less        according to maximum solubility of the drug),    -   ratios of activated charcoal/antibiotic of 3/1 and 10/1 were        tested,    -   incubation for 2 h at 37° C. (15 ml polypropylene tube, slow        rotation 20 rpm),    -   sampling at 0.5 h, 1 h and 2 h,    -   Residual antibiotic dosage (i.e., not adsorbed) was determined        by UV/visible spectrophotometric analysis.

Results are shown in tables 1 and 2.

TABLE 1 in vitro adsorption of various antibiotics usually administeredto humans Initial amount of Residual Residual Residual Residual ATBantibiotic antibiotic antibiotic antibiotic Antibiotic tested (μg/ml)after 30 min after 2 h (*) after 30 min after 2 h (*) Ratio of activated3/1 6/1 after 1 h 10/1 20/1 after 1 h charcoal/ATB and and 9/1 after 2 h30/1 after 2 h Beta-lactams Amoxicillin 200 19%  5% Ampicillin 200 15%<1% Piperacillin 200 <1% <1% Cephalexine 50 26% <10%  Cefuroxime 50  5%<5% Ceftriaxone 200  1% <1% Cefotaxime 200 <1% <1% Ceftiofur 50 <5% <5%Cefixime 50 10% <2.5%   Cefdinir 50  9% <1% Cefpodoxime 50 13% <3%Cefquinome 50 <5% <5% Cefepime 50  9% <5% Imipeneme 200  6% <2.5%  Ertapeneme 50 20% <5% Clavulanate 50 73% 22% 39% 3.5% Tazobactam 50 36%<20%  <20%  Tetracyclines Chlortetracycline 50 <3% <3% Oxytetracycline200 <2.5%   <2.5%   Tetracycline 200 <0.1%   <0.1%   Doxycycline 200<0.1%   <0.1%   Minocycline 200 <0.3%   <0.3%   Macrolides Tylosine 20066% <3%  6%  <3% Erythromycin 200 49% <0.3%   Azithromycin 20 78%  1%Clarithromycin 200 54% <0.3%   Fluoroquinolones Ciprofloxacine 50  2%Levofloxacine 50  1% <1% Marbofloxacine 200 <1.25%   <1.25%   SulfamidesSulfamethoxazole 50  5% <5% Trimetoprime 50 11% <3% Linezolide 200<1.25%   <1.25%   Glycopeptides Vancomycin 200 81% 27% 53% <1.25% Others Florfenicol 200  8% <2.5%   Tiamulin 200 77.66%   71.66%  Tigecycline 200 37% 21% <2.5%   (*) If antibiotic is not fully adsorbedafter 30 min at 3/1 or 10/1 ratio, then extra charcoal is added after 1and 2 hours ATB is an abbreviation for antibiotic

TABLE 2 ex vivo adsorption of various antibiotics usually administeredto humans Initial Amount of ATB amount of % of % of found in human Doseantibiotic residual residual feces (literature) administered Antibioticadded antibiotic antibiotic mean ± SD range and route of tested (μg/ml)after 5 h after 5 h (μg/g) (μg/g) administration Reference Ratio of 10/150/1 activated charcoal/ATB Beta-lactams Amoxicillin ND 500 mgX3 oralInternal data Piperacillin  0-276 4 g i.v. (1) Cefuroxime ND 250 mgX2oral Internal data Ceftriaxone 152 ± 53   0-657 2 gX1 i.v. (2) Cefdinir200 0.2%  0.15%  Cefpodoxime 200  2%  5% 550 ± 460 95-550 200 mgX2 oralInternal data Imipeneme 0.7-11.3 500 mgX4 i.v. (3) Ertapeneme 37.2 ±110   0-330 1 gX1 i.v. (3) Clavulanate Tazobactam 0.8-22.2 4 g i.v. (4)Tetracyclines Tétracycline 200 2.8%   0% Macrolides Erythromycin 200 12%0.3% 978 ± 219 1 gX2 oral (5) Azithromycin 196 17-510 Clarithromycin127.8 ± 58   500 mgX2 oral (5) Fluoroquinolones Ciprofloxacine 50 18%0.2% Levofloxacine 50  8% 87.4 ± 59.5 500 mgX1 oral (6) OxazolidonesLinezolide 200 13% 6.5% ND = Not detectable (1) Nord, C. E., et al.“Effect of piperacillin/tazobactam treatment on human bowel microflora.”J. Antimicrob. Chemother. 31 Suppl A (1993): 61-65. (2) Pletz, M. W., etal. “Ertapenem pharmacokinetics and impact on intestinal microflora, incomparison to those of ceftriaxone, after multiple dosing in male andfemale volunteers.” Antimicrob. AgentsChemother. 48.10 (2004): 3765-72.(Pletz et al. 3765-72) (3) Kager, L., et al. “Effect of imipenemtreatment versus imipenem surgical prophylaxis on the intestinalmicroflora.” Int. J. Clin. Pharmacol. Res. 8.6 (1988): 441-47. (4)Brismar, B., C. Edlund, and C. E. Nord. “Comparative effects ofclarithromycin and erythromycin on the normal intestinal microflora.”Scand. J. Infect. Dis. 23.5 (1991): 635-42. (Brismar, Edlund, and Nord635-42) (5) Edlund, C., et al. “Comparative effects of moxifloxacin andclarithromycin on the normal intestinal microflora.” Scand. J. Infect.Dis. 32.1 (2000): 81-85. (Edlund et al. 81-85) (6) Edlund, C., S.Sjostedt, and C. E. Nord. “Comparative effects of levofloxacin andofloxacin on the normal oral and intestinal microflora.” Scand. J.Infect. Dis. 29.4 (1997): 383-86. (Edlund, Sjostedt, and Nord 383-86)

As shown in the tables above, most of the antibiotics tested can beadsorbed significantly onto activated charcoal, at a ratio that can beextrapolated in human as clinically relevant. In vitro data correlatewell with ex vivo data and where data is available from literature, itcan be seen that the amount of residual antibiotic found into the fecescan be easily removed with activated charcoal formulation.

Example 7: Pharmaceutical Formulation

The feasibility of an oral dosage form for the site specific delivery ofactivated charcoal was investigated by testing different pharmaceuticalformulation processes. The objective was to develop a galenic formappropriate to the delayed release of activated charcoal in the laterpart of the gastrointestinal tract yet preserving as much as possiblethe adsorption characteristics of the charcoal.

Activated charcoal is a very challenging product to formulate because ofits physicochemical properties such as low density, hydrophobicity,wetting properties, etc. Attempting to formulate the charcoal for theintended use described in this invention at a therapeutic dose for humanadministration was not possible using conventional direct compressionbecause of the very low cohesive properties of activated charcoal. Evensimple wet granulation and compression lead to tablets exhibiting pooradsorption properties. The inventors, however, managed to formulateactivated charcoal in large amounts in delivery systems that elicit verygood stability and disintegration properties: a fast and efficientdispersion of activated charcoal in solution is obtained. Furthermore,the adsorption properties of activated charcoal are preserved in thedescribed formulation.

Table below shows one example of pellets obtained by wet granulationfollowed by extrusion spheronization.

TABLE 3 example of a charcoal pellet formulation obtained by wetgranulation with carrageenan. Formulation Amount (%) Activated charcoal85 Gelcarin GP911 (κ-carrageenan) 15 Water Sufficient amount for 100%

These pellets constitute the core of the formulations used throughoutthe present examples.

These pellets are then further coated with specific pH-dependent polymercoating, such as Eudragit® FS30D or Eudragit® L30D55 (Evonik, Darmstadt,Germany) for example.

The capacity of formulated and non formulated activated charcoal toadsorb various antibiotics in simulated colonic conditions was studiedin adsorption kinetic studies.

For this purpose, coated pellets of activated charcoal were firstdeformulated in a buffer (50 mM sodium phosphate buffer, 80 mM NaCl, pH7.5) for at least 30 minutes at 37° C. Suspensions of non formulatedactivated charcoal (NFAC) were also prepared in this buffer. Then, theadsorption capacity of a suspension of deformulated pellets(formulation) and a suspension of NFAC were tested with a levofloxacinsolution (levo).

FIG. 6 presents, as an example, a comparison of several adsorptionkinetics of levofloxacin on NFAC and deformulated pellets. Theexperiments were performed as in the previous example, with pelletscoated with 20% Eudragit FS30D.

Two studied ratios of NFAC to levofloxacin and deformulated pellets tolevofloxacin are presented, 3:1 and 10:1. A control sample, made of thesolution of levofloxacin, was also analyzed during the adsorptionkinetic.

Full adsorption of levofloxacin on deformulated pellets and NFAC isobserved after 60 minutes for the 10:1 ratio. The adsorption is almostcomplete for the ratio 3:1 on the formulation and complete on NFAC.Hence, the adsorption properties of the activated charcoal areessentially maintained through the formulation processes.

Limited influence of time, temperature and humidity could be observed inbulk storage conditions of the formulation. Excellent stability wasobtained at room temperature after 9 months in bulk storage conditionsas determined by measurement of disintegration time and ciprofloxacinadsorption after one hour.

More precisely, stored pellets were disintegrated in the simulatedcolonic medium (50 mM sodium phosphate buffer, 80 mM NaCl, pH 7.5) andspiked with a known amount of ciprofloxacin. The kinetics ofdisintegration of the pellets was monitored. As pellets disintegrate andrelease the activated charcoal, the ciprofloxacin concentration of thesolution decreases. The results presented in Table 4 below represent theremaining percentage of ciprofloxacin in the media at different samplingtimes. These results prove that the disintegration properties of thepellets are maintained during 9 months in bulk storage conditions.

TABLE 4 Bulk stability of FS30D-coated charcoal formulation stored atroom temperature in glass vials. Charcoal formulation/ ciprofloxacinratio was 9:1. Results are expressed as the percentage of freeciprofloxacin remaining in the solution. Time (min) T0 T6 months T9months 0 95 99 100 30 49 51 85 60 2 4 7 120 <1 2 2 180 <1 <1 <1

Example 8: In Vitro Release Profile of Activated Charcoal and AdsorptionKinetics of Ciprofloxacin

One of the major issues with charcoal formulation is the disintegrationprofile of the charcoal pellet into the medium to allow maximumadsorption efficiency. The formulation described earlier has been testedin a BioDis dissolution tester (USP III release apparatus) using severalsimulated intestinal media spiked with ciprofloxacin at a concentrationof 50 μg/mL. In this experiment, approx. 73 mg of coated pellets weresubmitted to dissolution in the BioDis system, by being successivelyincubated for the indicated times in media whose composition reflect thepH, buffer capacity and osmolarity of the various gastro-intestinalcompartments. Samples of each medium were withdrawn and analyzed todetermine the remaining ciprofloxacin concentration. Ciprofloxacin wasonly adsorbed by the active charcoal released from the formulation,hence ciprofloxacin adsorption was taken as a proxy or active charcoalrelease from the formulation.

The simulated gastro-intestinal media are described below:

Simulated gastric medium: 34.2 mM NaCl, pH adjusted to 1.6 with HCl.

Simulated duodenum and proximal jejenunum medium: 19.1 mM maleic acid,70 mM NaCl, 31.6 mM NaOH, pH=6.5

Simulated middle and late jejunum medium: 25 mM HEPES, 121.6 mM NaCl, pHadjusted to 7.0

Simulated ileal medium: 18 mM HEPES, 132.1 mM NaCl, pH adjusted to 7.5

Simulated colonic medium: 18.7 mM maleic acid, 83.7 mM NaCl, 25.6 mMNaOH, pH adjusted to 6.0

A shown in FIG. 7, the carrageenan-based charcoal formulation coatedwith FS30D, as a coating example, demonstrated no charcoal release untilreaching pH of 7.5. Then, adsorption of ciprofloxacin was very fast andcomplete within half an hour after charcoal dispersion.

Example 9: Other Possible Formulations

Antibiotics have different absorption profiles in mammals, some beingabsorbed early and some being absorbed later. The later will reach theirmaximum plasma concentration after 2 to 4 hours. Formulations can bedeveloped to allow a more delayed adsorbent delivery to avoid any impacton the normal absorption process of the antibiotic.

To achieve such a delayed release, pellet formulations were first coatedwith a sub-coat that prevented them from disintegrating too fast anddelayed the disintegration by 30 minutes to 2 hours, depending on thetype of polymer used for the sub-coat.

Various formulations were achieved using multiple coating techniques.Table 5 below presents some examples of coating combinations

TABLE 5 Formulations with various sub-coats and final coating with FS30DSubCoat details (w/w Outer final Coating SubCoat type weight) OuterCoating type amount L30D55/NE30D(2/8)*   15% — — L30D55/NE30D(2/8)*  25% — — L30D55/NE30D(2/8)*   35% — — L30D55/NE30D(2/8)* 12.75% FS30D15% L30D55/NE30D(2/8)* 21.25% FS30D 15% L30D55/NE30D(2/8)* 29.75% FS30D15% None None Aqoat HF 15% None None Aqoat HF 20% None None Aqoat HF 25%None None Aqoat HF 30% None None Aqoat HF 35% None None Shellac Aquagold15% None None Shellac Aquagold 20% None None Shellac Aquagold 25% NoneNone Shellac Aquagold 30% None None Shellac Aquagold 35% None NoneEthylcellulose 2% None None Ethylcellulose 4% None None FS30D/L30D55(9/1)* 15% None None FS30D/L30D55 (9/1)* 20% None None FS30D/L30D55(9/1)* 25% None None FS30D/L30D55 (85/15)* 15% None None FS30D/L30D55(85/15)* 20% None None FS30D/L30D55 (85/15)* 25% None None FS30D 15%None None FS30D 35% *the numbers in parentheses represent the respectiveproportions of the indicated Eudragit polymers in the mixture used toprepare the pellet subcoat or outer coat.

Dissolution tests were performed on these pellets in simulated ilealmedium, pH7.5 to assess the kinetics of pellet dispersion in such amedium and compare the obtained delays. The ileum medium was spiked with50 μg/mL ciprofloxacin, and at each sampling time, the amount ofresidual ciprofloxacin remaining in the solution was quantified;ciprofloxacin adsorption was taken as a proxy for charcoal release.

BioDis tests were also performed on these pellets, as described above,in order to mimic their progression through the gastro intestinal tract.This enabled a more detailed characterization of the delayed release ofcharcoal from the pellets.

FIGS. 8-10 describe the charcoal release, measured as ciprofloxacinadsorption, from some of the formulations described in Table 5.

As can be seen in FIG. 8, a first coating (subcoat) made of a mixtureL30D55/NE30D polymers representing 35% w/w of the final weight of thepellets, elicited a delayed adsorption of ciprofloxacin, meaning thatcharcoal exhibited a delayed release of charcoal as compared to theformulation made with an outer FS30D coat by approx. 30 minutes.

As can be seen in FIG. 9, the Aqoat-coated pellets exhibited adissolution that was approx 30 min faster than those coated with anequivalent amount of FS30D. Ciprofloxacin adsorption was complete withinone hour. For pellets coated with Shellac, delayed dissolution wasobserved for the film and disintegration of the charcoal pellets wasprolonged for at least two hours

As can be seen in FIG. 10, the effect of various thicknesses ofethylcellulose coatings was assessed on pellet dissolution. Anintermediate coat consisting in 2% ethylcellulose induced a significantdelayed release of activated charcoal, by approximately 40 minutes toone hour, compared to 20% FS30D coating.

Such formulations might be of interest in providing delayed andprolonged release of the adsorbent.

Example 10: In Vitro Adsorption Kinetics of Irinotecan and SN-38 ontoActivated Charcoal

The adsorption kinetics of irinotecan and its active metabolite SN-38onto active charcoal were determined in vitro (FIGS. 11A and 11Brespectively).

The capacity of activated charcoal to adsorb irinotecan (200 μg/mLinitial concentration) was assessed in simulated ileal medium (18 mMHEPES, 132.1 mM NaCl, adjusted to pH 7.5 with NaOH). The respectiveproportions of activated charcoal and irinotecan were 3:1 and 10:1, inrelation to irinotecan. Samples were centrifuged, filtered, and dilutedten-fold, prior to determination of the non-adsorbed concentration ofirinotecan by measuring its absorbance at 368 nm using aspectrophotometer. As can be seen in FIG. 11A, half of the amount ofirinotecan was adsorbed in about 12 minutes with a 3:1 ratio ofactivated charcoal/irinotecan. Complete adsorption was achieved in about15 minutes with a 10:1 ratio of activated charcoal/irinotecan.

FIG. 11B shows the capacity of activated charcoal to adsorb SN-38. SN-38was dissolved at a concentration of 50 μg/mL in 0.01 M NaOH pH 12. Nonadsorbed SN-38 was detected after centrifugation and filtration by atits absorbance at 411 nm using a spectrophotometer. As can be seen inFIG. 11B, SN38 adsorption was complete in about than 30 minutes with anactivated charcoal/SN-38 ratio of 10:1.

The adsorption kinetics of irinotecan and its metabolite SN-38 ontoactivated charcoal (NFAC) (FIG. 11C) and charcoal released fromdeformulated coated pellets (DCP) (20% of FS30D coating) (FIG. 11D) weredetermined ex vivo, in piglet caecal medium.

Piglet caecal medium was spiked with 250 μg/ml irinotecan and 50 μg/mlSN-38, and preincubated for 2 h at 37° C. NFAC or DCP was pre-incubatedwith piglet caecal medium diluted 1:1 for 2 hours at 37° C. The ratiosof NFAC/irinotecan were 10:1, 50:1, and those for NFAC/SN38 were 10:1,50:1, 100:1 and 250:1; the ratios of active charcoal from DCP toirinotecan and SN38 were 10:1 and 50:1.

After combining these two pre-incubation mixed, incubation was carriedout at 37° C. with gentle agitation for the indicated periods of time.Samples were withdrawn, centrifuged, the supernatant was filtered andanalysed for the presence of irinotecan and SN38 by HPLC.

Example 11: In Vivo Performance of Targeted-Release Activated Charcoalin Reducing the Emergence of Bacterial Resistance to Antibiotic

A proof of concept (POC) study of the ability of targeted-releaseactivated charcoal (coated pellets of activated charcoal) to reduce theemergence of bacterial resistance during antibiotic treatments wasperformed in piglets, that were weaned 4 weeks after birth, and includedinto the study two weeks thereafter.

The study was randomized, comparative, open for in-life phase butblinded to treatment for evaluation of microbiological and PK/PD data todemonstrate the efficacy of charcoal delayed-release formulation to:

-   -   Decrease antibiotic fecal concentrations    -   Prevent the emergence of bacterial resistance in gut flora.    -   Maintain the normal antibiotic absorption process.

To carry out this in vivo POC, the antibiotic to be tested wasciprofloxacin, a fluoroquinolone, administered orally at a dose of 1.5mg/kg/day. This type of study will be applicable for any antibioticconsidered by Da Volterra. The methods used in evaluating the decreaseof antibiotic fecal concentrations and the emergence of bacterialresistance were developed by Da Volterra.

The experiments were conducted under GLP conditions. Piglets, from thesame batch, had not been treated with antibiotics since birth.

The study design is depicted in FIG. 12.

The primary endpoints of this study were:

Pharmacokinetic Criteria:

-   -   To compare the concentration of ciprofloxacin with or without        coated pellets in feces by the comparison of Neperian logarithm        of the Area Under the Curve (AUC) of fecal ciprofloxacin        concentrations between Day 1 and Day 9 (log AUC_(D1-D9)).    -   The AUC between Day 1 and Day 9 were computed by trapezoidal        approach using SAS software and are analyzed with descriptive        statistics and compared across groups by t-tests (on log        AUC_(D1-D9)).    -   To compare the plasma concentrations of ciprofloxacin with or        without coated pellets by comparing        -   Neperian logarithm of Area Under the Curve (AUC) of            ciprofloxacin plasma concentrations between 0 h and the time            T_(last) corresponding to the last observed value (log AUC)            (This choice of AUC_(0-last) can be explained by the very            high percentages of extrapolation for AUC_(0-∞))        -   Neperian logarithm of ciprofloxacin plasma maximum            concentration (Cmax) (log Cmax)

This was performed by a Non Compartmental Approach (NCA), linear/logtrapezoidal method, via WinNonLin software (version 5.2) to computeC_(max) and AUC. Ciprofloxacin plasma concentrations at 0 h wereconsidered to be 0 ng/mL. Log AUC and log C_(max) were then calculatedand analyzed with descriptive statistics.

Pharmacodynamics Criterion:

To compare the number of resistant bacteria after treatment byciprofloxacin with or without coated pellets by comparing the AUC ofcounts of Enterobacteriaceae resistant to ciprofloxacin, and tonalidixic acid, from Day 1 to Day 6 (treatment) normalized to Day −1/1.The bacterial counts were obtained by performing 100 μl of 1/10 dilutionof feces plated on Drigaski agar. The counts of Enterobacteriaceaeresistant to ciprofloxacin and nalidixic acid were obtained by platingdiluted feces on Drigaski agar with 2 ml/l ciprofloxacin and 20 ml/lnalidixic acid. The detectable limit of resistant Enterobacteriaceaecounts was 1.00×10² CFU/g. The baseline was calculated on the meancontent of resistant bacteria before treatment and area under the curveis the area between the baseline and the curve of log 10 ofciprofloxacin resistant Enterobacteriaceae counts.

Results

Coated pellets associated to ciprofloxacin was able to decrease theresidual fecal concentrations of ciprofloxacin in piglets. The decreasewas statistically significant. The comparative results of ciprofloxacinconcentration in feces are showed in FIG. 13 and there are summarized inTable 6.

TABLE 6 Fecal ciprofloxacin concentrations: descriptive statistics onindividual log AUC D1-D9 by group (n1 = 6, n2 = 11, n3 = 12) AnalysisVariable: log AUC_(D1-D9) Group N Mean Median SD Minimum Maximum Group 16 −0.24 0.00 0.59 −1.37 0.28 (placebo/ placebo) Group 2 11 4.04 4.310.53 3.21 4.64 (ciprofloxacin/ placebo) Group 3 12 2.98 3.21 0.53 2.033.62 (ciprofloxacin/ coated pellets)

The difference of log AUC_(D1-D9) between Group 2(ciprofloxacin/placebo) vs. Group 3 (ciprofloxacin/coated pellets) wasstatistically significant by comparison t-test (p-value<0.0001).

There were also significant differences between Group 1(placebo/placebo) vs. Group 2 (ciprofloxacin/placebo) (p-value<0.0001)and between Group 1 (placebo/placebo) vs. Group 3(ciprofloxacin/Dav-132) (p-value<0.0001).

Coated pellets administration associated to ciprofloxacin administrationdid not result in a significant change in ciprofloxacin plasmaconcentration. The results on ciprofloxacin concentration in plasma areshowed in FIG. 14 and they are summarized in Table 7.

TABLE 7 Plasma concentrations of ciprofloxacin: descriptive statisticson individual log AUC and log Cmax by group (n2 = n3 = 12) Group N MeanMedian SD Min Max Analysis Variable: log AUC Group 2 12 6.82 6.79 0.615.68 7.88 (ciprofloxacin/ placebo) Group 3 12 6.54 6.46 0.62 5.50 7.52(ciprofloxacin/ coated pellets) Analysis Variable: log Cmax Group 2 125.48 5.44 0.71 4.18 6.64 (ciprofloxacin/ placebo) Group 3 12 5.27 5.130.83 4.12 6.93 (ciprofloxacin/ coated pellets)

The results show that there is no significant difference between these 2groups on log AUC (t-test p-value=0.28) and on log Cmax (t-testp-value=0.51). Administration of coated pellets together withciprofloxacin resulted in a decrease in bacterial resistance due to theresidual concentration of ciprofloxacin in feces (see FIG. 15).

Ciprofloxacin-resistant Enterobacteriaceae counts in faeces increasesignificantly with the treatment of piglets with ciprofloxacin. Charcoalformulations according to the invention administered with ciprofloxacinreduced the emergence of bacterial resistance significantly as showed inTable 8. The control group (placebo/placebo) showed no emergence ofresistance.

TABLE 8 Resistant bacterial counts: descriptive statistics of individualAUC between Day 1 and Day 6 for ciprofloxacin and nalidixic acid T-testsGroup ciprofloxacin/coated pellets Criteria vs. ciprofloxacin/placeboAUC_(cipro D1-D6) 0.0430 AUC_(nal D1-D6) 0.0478Conclusions

The results showed that the formulations according to the inventionwere:

-   -   Well tolerated by piglets    -   Able to reduce significantly the concentration of ciprofloxacin        in the faeces after administration for a five day period        together with oral ciproflixacin    -   Able to significantly reduce the emergence of bacterial        resistance to antibiotic treatment    -   Able to show no interference with the normal process of        ciprofloxacin absorption

Example 12: In Vitro Adsorption Kinetics of Pancreatic Enzymes ontoActivated Charcoal

The adsorption kinetics of Creon, a medicine containing pancreaticenzymes, onto active charcoal were determined in vitro (FIG. 16). Theextent of enzyme adsorption onto activated charcoal was assessed using aprotein quantification assay (Bradford method). The capacity ofactivated charcoal to adsorb pancreatic enzymes (1 mg/mL of Creon) wasassessed in buffer (50 mM sodium phosphate buffer, 80 mM NaCl, adjustedto pH 7.5). The respective proportions of activated charcoal and Creonwere 9:1, 15:1 and 25:1. Samples were centrifuged, the supernatant wasfiltered, and the amount of residual protein was quantified using aBradford protein assay. A solution of 1 mg/ml Creon, and a 3 mg/mlsuspension of activated charcoal were respectively used as positive andnegative controls. As can be seen in FIG. 16, complete adsorption of theenzymes was obtained in 2 hours with a 15:1 ratio and in 1 hour with a25:1 ratio.

The invention claimed is:
 1. A formulation comprising: a) a substantially water-free core comprising a pellet of activated charcoal mixed with carrageenan, wherein the amount of carrageenan is between 5% and 25% by weight of the mixture of the activated charcoal with the carrageenan and wherein the amount of activated charcoal is between 75%-95% by weight of the mixture of the activated charcoal with the carrageenan, and b) a layer of an external coating formed around the core, wherein the external coating is a pH-dependent enterosoluble polymer that dissolves at a pH equal to 6.0 and above.
 2. The formulation of claim 1, wherein the amount of carrageenan is about 15% by weight of the mixture of the activated charcoal with the carrageenan.
 3. The formulation of claim 1, wherein a further coating is provided between the core and the external pH-dependent layer, and wherein said further coating is selected from the group consisting of pH-dependent polymers, pH-independent water soluble polymers, pH-independent water insoluble polymers, and mixtures of a pH-dependent polymer and a water insoluble, pH-independent polymer.
 4. The formulation of claim 3, wherein the pH-independent water soluble polymer is selected from the group consisting of polyvinyl pyrrolidone (PVP) and cellulose polymers.
 5. The formulation of claim 4, wherein the cellulose polymers are selected from the group consisting of hydroxypropylmethylcellulose (HPMC) and hydroxypropylcellulose (HPC).
 6. The formulation of claim 5, wherein the pH-independent water insoluble polymers are selected from the group consisting of ethylcellulose polymers and copolymers of ethyl acrylate and methyl methacrylate.
 7. The formulation of claim 3, wherein the further coating comprises ethylcellulose or ethyl acrylate methyl methacrylate copolymer.
 8. The formulation according to claim 3, wherein the further coating comprises at least one cellulose derivative selected from the group consisting of hydroxypropylcellulose and ethylcellulose.
 9. The formulation according to claim 3, wherein the further coating is made of a 1:9 to 9:1 weight ratio mixture of a methacrylic acid and ethyl acrylate copolymer and an ethyl acrylate and methyl methacrylate copolymer.
 10. The formulation of claim 1, wherein the enterosoluble polymer layer is from 10% to 40% by weight of the total formulation.
 11. The formulation of claim 1, wherein the activated charcoal is USP grade activated charcoal.
 12. The formulation of claim 1, wherein the activated charcoal has a surface area above 1500 m²/g.
 13. The formulation of claim 1, wherein the carrageenan is kappa-carrageenan.
 14. A formulation comprising: a) a core comprising activated charcoal mixed with carrageenan, wherein the amount of carrageenan is between 5% and 25% by weight of the mixture of the activated charcoal with the carrageenan and wherein the amount of activated charcoal is between 75%-95% by weight of the mixture of the activated charcoal with the carrageenan, and b) a layer of an external coating formed around the core that dissolves at a pH equal to 6.0 and above.
 15. The formulation of claim 14, wherein the carrageenan is kappa-carrageenan.
 16. The formulation of claim 14, wherein the amount of carrageenan is about 15% by weight of the mixture of the activated charcoal with the carrageenan.
 17. A formulation comprising: a) a core comprising a pellet of activated charcoal mixed with carrageenan, wherein the amount of carrageenan is between 5% and 25% by weight of the mixture of the activated charcoal with the carrageenan and wherein the amount of activated charcoal is between 75%-95% by weight of the mixture of the activated charcoal with the carrageenan, and b) a layer of an external coating formed around the core, wherein the external coating is a pH-dependent enterosoluble polymer that dissolves at a pH equal to 6.0 and above.
 18. The formulation of claim 17, wherein the carrageenan is kappa-carrageenan.
 19. The formulation according to claim 1, wherein the pH-dependent enterosoluble polymer is selected from the group consisting of anionic copolymers based on methylacrylate, methylmethacrylate and methacrylic acid.
 20. The formulation according to claim 14, wherein the pH-dependent enterosoluble polymer is selected from the group consisting of anionic copolymers based on methylacrylate, methylmethacrylate and methacrylic acid.
 21. The formulation according to claim 17, wherein the pH-dependent enterosoluble polymer is selected from the group consisting of anionic copolymers based on methylacrylate, methylmethacrylate and methacrylic acid. 